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Abstract:

The present invention relates to microfluidic devices that include a
reliable seal between a substrate of the device and a fluid transport
mechanism. The devices of the invention include at least one internal
channel, and at least one port in fluid communication with the channel. A
seal is associated with the port and is configured to receive a fluid
transport mechanism. The seal is formed from an elastomeric material that
is compatible for use with fluorinated oil and resists flaking and
degradation.

Claims:

1. A microfluidic device, comprising: a member defining at least one
internal channel and at least one port in fluid communication with each
of the channels; and a gasket associated with each of the ports and
configured to sealingly receive a fluid transport mechanism such that
said fluid exits the transport mechanism and enters one of the channels,
the gasket comprising an elastomeric material comprised of a two phase
block copolymer having a soft polydimethylsiloxane phase and a hard
aliphatic isocyanate phase.

7. The microfluidic device of claim 1 wherein a portion of the gasket
fits at least partially into the port and another portion of the gasket
sealingly receives the fluid transport mechanism.

8. The microfluidic device of claim 1 wherein the member comprises a top
plate adhered to a bottom plate.

9. The microfluidic device of claim 8 wherein each of the top and bottom
plates has a top surface and a bottom surface, and wherein the top
surface of the bottom plate faces and is adhered to the bottom surface of
the top plate.

10. The microfluidic device of claim 9 wherein the bottom plate defines
the channels and the top plate defines the ports.

11. The microfluidic device of claim 1, wherein the fluid transport
mechanism is a pipette or a tube.

12. A microfluidic device, comprising: a member defining at least three
internal channels and also defining a first inlet port and a first outlet
port of a first one of the channels, a second inlet port and a second
outlet port of a second one of the channels, and a third inlet port and a
third outlet port of a third one of the channels; a first gasket
associated with the first, second, and third inlet ports and configured
to sealingly receive a fluid input mechanism such that fluid exits the
fluid input mechanism and enters one of the first, second, and third
channels via one of the first, second, and third inlet ports; and a
second gasket associated with the first, second, and third outlet ports
and configured to sealingly receive a fluid output mechanism such that
fluid exits one of the first, second, and third channels via one of the
first, second, and third outlet ports and enters the fluid output
mechanism, wherein each of the first and second gaskets comprise an
elastomeric material comprised of a two phase block copolymer having a
soft polydimethylsiloxane phase and a hard aliphatic isocyanate phase.

13. The microfluidic device of claim 12, wherein the fluid input
mechanism is a pipette or a tubing.

14. The microfluidic device of claim 13, wherein the fluid output
mechanism is a pipette or a tubing.

15. The microfluidic device of claim 12 wherein each of the first and
second gaskets comprises a thermoplastic silicone elastomer.

16. The microfluidic device of claim 15 wherein each of the first and
second gaskets is formed by injection molding.

18. The microfluidic device of claim 12 wherein each of the first and
second gaskets is compatible with a fluorinated oil.

19. The microfluidic device of claim 12 wherein each of the first and
second gaskets resists flaking and degradation after sealingly receiving
the fluid input and fluid output mechanisms, respectively.

20. The microfluidic device of claim 12 wherein the first gasket includes
a first bottom portion that fits at least partially into the first inlet
port, a second bottom portion that fits at least partially into the
second inlet port, and a third bottom portion that fits at least
partially into the third inlet port.

21. The microfluidic device of claim 20 wherein the first gasket includes
a first top portion that sealingly receives the fluid input mechanism to
allow fluid that exits the fluid input mechanism to enter the first
channel, a second top portion that sealingly receives the fluid input
mechanism to allow fluid that exits the fluid input mechanism to enter
the second channel, and a third top portion that sealingly receives the
fluid input mechanism to allow fluid that exits the fluid input mechanism
to enter the third channel.

22. The microfluidic device of claim 12 wherein the second gasket
includes a first bottom portion that fits at least partially into the
first outlet port, a second bottom portion that fits at least partially
into the second outlet port, and a third bottom portion that fits at
least partially into the third outlet port.

23. The microfluidic device of claim 22 wherein the second gasket
includes a first top portion that sealingly receives the fluid output
mechanism to allow fluid that exits the first channel to enter the fluid
output mechanism, a second top portion that sealingly receives the fluid
output mechanism to allow fluid that exits the second channel to enter
the fluid output mechanism, and a third top portion that sealingly
receives the fluid output mechanism to allow fluid that exits the third
channel to enter the fluid output mechanism.

24. The microfluidic device of claim 12 wherein the member comprises a
top plate adhered to a bottom plate.

25. The microfluidic device of claim 24 wherein each of the top and
bottom plates has a top surface and a bottom surface, and wherein the top
surface of the bottom plate faces and is adhered to the bottom surface of
the top plate.

26. The microfluidic device of claim 25 wherein the bottom plate defines
the channels and the top plate defines the ports.

27. The microfluidic device of claim 12 further comprising a carrier into
which the microfluidic device is disposed.

28. A disposable cartridge for use with a microfluidic analysis system,
comprising: a carrier; and a microfluidic device disposed within the
carrier and comprising: a member defining at least three internal
channels and also defining a first inlet port and a first outlet port of
a first one of the channels, a second inlet port and a second outlet port
of a second one of the channels, and a third inlet port and a third
outlet port of a third one of the channels; a first gasket associated
with the first, second, and third inlet ports and configured to sealingly
receive an input pipette such that fluid exits a tip of the input pipette
and enters one of the first, second, and third channels via one of the
first, second, and third inlet ports; and a second gasket associated with
the first, second, and third outlet ports and configured to sealingly
receive an output pipette such that fluid exits one of the first, second,
and third channels via one of the first, second, and third outlet ports
and enters a tip of the output pipette, wherein each of the first and
second gaskets comprise an elastomeric material comprised of a two phase
block copolymer having a soft polydimethylsiloxane phase and a hard
aliphatic isocyanate phase.

Description:

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application
No. 61/437,491, filed Jan. 28, 2011, the contents of which are herein
incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present invention relates to microfluidic systems. More
specifically, the invention relates to gaskets for sealing fluid
interfaces in microfluidic systems.

BACKGROUND INFORMATION

[0003] Microfluidic devices are known. A microfluidic nozzle array device
disclosed in U.S. Pat. No. 6,800,849 uses an O-ring sealing gasket. U.S.
Pat. No. 7,390,463 also discloses the use of an O-ring, in connection
with each of a plurality of microfluidic modules that together form a
support structure or plate.

[0004] Precision manipulation of streams of fluids with microfluidic
devices is revolutionizing many fluid-based technologies. Networks of
small channels are a flexible platform for the precision manipulation of
small amounts of fluids. Virtually all microfluidic devices are based on
flows of streams of fluids. Current microfluidic technologies utilize
aqueous droplets in an immiscible carrier fluid. Such droplets provide a
well-defined, encapsulated microenvironment that eliminates cross
contamination or changes in concentration due to diffusion or surface
interactions. While significant advances have been made in dynamics at
the macro- or microfluidic scale, improved fluid handling technology is
still needed.

[0005] Microfluidic devices for performing biological, chemical, and
diagnostic assays are described in U.S. Published Patent Application No.
US2008/0003142 and US2008/0014589, each of which is incorporated by
reference herein in its entirety. Such microfluidic devices generally
include at least one substrate having one or more microfluidic channels
etched or molded into the substrate, and one or more interconnects (fluid
interface). The one or more interconnects contain inlet modules that lead
directly into the microfluidic channels, and serve to connect the
microfluidic channel to a means for introducing a sample fluid to the
channel. The one or more interconnects also serve to form a seal between
the microfluidic substrate and the means for introducing a sample. The
one or more interconnects can be molded directly into the microfluidic
substrate, as one or more individual pieces, or as a single, monolithic
self-aligning piece (see e.g., FIGS. 11-13 of US2008/0003142, herein
incorporated by reference in its entirety).

[0006] For pressure-driven microfluidic chips, it is essential to
establish a reliable fluid interface. The mechanism(s) employed to
introduce a sample fluid into the microfluidic channel, such as tubing or
pipettes, is typically inserted in a simple linear motion, and it is
important that a reliable seal be established in the first attempt to
avoid sample contamination. The seal must be able to withstand and hold a
pressure of at least 70 psi, the minimum pressure in most pressure-driven
microfluidic devices. Furthermore, the seal component(s) must be suitable
for use with both water and oil based fluids, given the trend in droplet
technology towards the use of aqueous droplets in an immiscible carrier
fluid (e.g., fluorinated oil).

SUMMARY

[0007] The present invention relates to a microfluidic device that
provides a reliable seal between a substrate of the device and the fluid
transport mechanism which typically will be one or more pipettes, tubing,
or other conduit providing a channel outside the microfluidic device. As
used herein, the term "pipette(s)" is not intended to encompass only
devices which require suction to draw fluids into them. Rather, the term
"pipette(s)", as used herein, includes any fluid carrier/conduit that is
configured to carry a discrete amount of fluid for depositing into a
microfluidic device. In particular, the present invention provides a
microfluidic chip that utilizes a gasket at the fluid interface to the
chip.

[0008] In one aspect, microfluidic chips according to the invention
include a substrate member defining at least one internal channel and at
least one port in fluid communication with the channels. In one
particular embodiment, the substrate member includes a top plate adhered
to a bottom plate to form the substrate with the channel(s) and port(s).
The top and bottom plates each include a top surface and a bottom
surface. The top surface of the bottom plate faces and is adhered to the
bottom surface of the top plate. The top plate can include the port(s),
and the bottom plate can include the channel(s), such that when these two
plates are brought together and adhered to each other the combination
forms the substrate with the channel(s) and the port(s). Alternatively,
the top plate can include the channel(s), and the bottom plate can
include the port(s), such that when these two plates are brought together
and adhered to each other the combination forms the substrate with the
channel(s) and the port(s). Microfluidic chips of the invention further
include an elastomeric gasket associated with each of the ports and
configured to sealingly receive a fluid transport mechanism (e.g., a
pipette or a tubing) into the port, such that fluid from the fluid
transport mechanism enters the channel via the port that leads to that
channel. At least a portion of the gasket fits at least partially into
the port, while another portion of the gasket sealingly receives the
fluid transport mechanism. When the fluid transport mechanism contacts
the gasket, that contact creates radial compression against the gasket to
form a fluid-tight seal against the port.

[0009] Preferably, the gasket is made from a material suitable for use
with a fluorinated oil, and that resists flaking and degradation after
sealingly receiving the fluid transport mechanism. In certain aspects,
the gasket is made from a thermoplastic silicone elastomer, for example,
by injection molding. In a particular embodiment, the gasket is made from
Genomier® 200.

[0010] The elastomeric gaskets are capable of establishing a fluid-tight
seal by the simple linear motion of a pipette being placed into contact
with the gasket. The radial compression caused by insertion of the
pipette into the gasketed port (and/or the chip with its gasketed port
can be moved toward the pipette) is sufficient to seal the gasket against
the port and allow fluid to exit the pipette and enter the channel
without any leakage of the fluid (or the fluid can be pulled from the
channel and into the pipette, also without any leakage of fluid). The
seal created by the elastomeric gaskets described herein can withstand
and hold pressure up to 100 psi, thereby providing a tight and complete
seal which eliminates, or at least significantly minimizes, the risk of
contamination of the sample fluid.

[0011] In another aspect, microfluidic chips according to the invention
include a substrate member defining at least three internal channels and
also defining a first inlet port and first outlet port of a first one of
the channels, a second inlet port and a second outlet port of a second
one of the channels, and a third inlet port and a third outlet port of a
third one of the channels. The substrate member includes a top plate
adhered to a bottom plate to form the substrate with the channel(s) and
port(s). The top and bottom plates each include a top surface and a
bottom surface. The top surface of the bottom plate faces and is adhered
to the bottom surface of the top plate. The top plate can include the
port(s), and the bottom plate can include the channel(s), such that when
these two plates are brought together and adhered to each other the
combination forms the substrate with the channel(s) and the port(s).
Alternatively, the top plate can include the channel(s), and the bottom
plate can include the port(s), such that when these two plates are
brought together and adhered to each other the combination forms the
substrate with the channel(s) and the port(s).

[0012] The microfluidic chip further includes a first gasket associated
with the first, second and third inlet ports and configured to sealingly
receive a fluid input mechanism (e.g., a pipette or tubing) such that
fluid from the fluid input mechanism enters one of the first, second and
third channels via one of the first, second and third inlet ports, and a
second gasket associated with the first, second and third outlet ports
and configured to sealingly receive a fluid output mechanism (e.g., a
pipette or tubing) such that fluid exits one of the first, second and
third channels via one of the first, second and third outlet ports and
enters the fluid output mechanism.

[0013] The first gasket includes a first, a second, and a third bottom
portion, each of which fits at least partially into the first, second and
third inlet ports, respectively. The first gasket further includes a
first, a second and a third top portion, each of which sealingly receives
the fluid input mechanism to allow fluid that exits the fluid input
mechanism to enter the first, second and third channels, respectively.

[0014] The second gasket includes a first, a second, and a third bottom
portion, each of which fits at least partially into the first, second and
third outlet ports, respectively. The second gasket further includes a
first, a second and a third top portion, each of which sealingly receives
the fluid output mechanism to allow fluids that exit the first, second
and third channels to enter the fluid output mechanism.

[0015] The first gasket, the second gasket, or both, are preferably made
from a material that is suitable for use with a fluorinated oil and
resists flaking and degradation after sealingly receiving the fluid input
and output mechanisms. In certain aspects, the first and/or second
gaskets are made from a thermoplastic silicone elastomer, for example, by
injection molding. In a particular embodiment, at least a portion of the
first and/or second gaskets are made from Genomier® 200.

[0016] In certain aspects, the microfluidic chips of the invention are
housed within a carrier.

[0017] These and other aspects of the invention are described in further
detail in the Figures and Detailed Description below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Non-limiting embodiments of the present invention will be described
by way of example with reference to the accompanying drawings, which are
schematic and are not intended to be drawn to scale. In the drawings,
each identical or nearly identical component illustrated is typically
represented by a single numeral. For the purposes of clarity, not every
component is labeled in every drawing, nor is every component of each
embodiment of the invention shown where illustration is not necessary to
allow those of ordinary skill in the art to understand the invention. In
the drawings:

[0020]FIG. 2 depicts a cross-section of a microfluidic chip/plate that
includes one or more port structures that include a tapered lead directly
in a microfluidic channel (reference 1), and a gasket that contains
matching tapered bosses configured to fit within the port structures
(reference 2).

[0021]FIG. 3 depicts the assembly of the gasket and fluidic chip/plate
shown in FIG. 2.

[0022]FIG. 4 depicts the use of a pipette tip to position and seal the
gasket depicted in FIGS. 2 and 3 within the port.

[0023]FIG. 5 is a general schematic of an exemplary embodiment of a
fluidic chip according to the invention, showing the general location of
various port modules for use with a microfluidic chip according to the
invention; FIGS. 5A and 5B depict cross-sections of the fluidic chip
shown in FIG. 5A; FIGS. 5C-5H depict enlarged details of the different
port modules shown in FIGS. 5A and 5B.

[0024]FIG. 6A depicts a cross-section of a microfluidic chip according to
the invention that includes a top plate and a bottom plate, and a gasket
overmolded directly onto the top plate; FIG. 6B depicts a
three-dimensional perspective of the various layers and components
contained within a microfluidic chip that includes one or more gaskets
overmolded directly onto the top plate

[0025]FIG. 7A depicts a three-dimensional perspective of the various
layers and components contained within a microfluidic chip that includes
one or more gaskets molded into pockets within the top plate; FIG. 7B
depicts an exploded view of the microfluidic chip of FIG. 7A, showing a
top plate with pockets, the gaskets that are molded into the pockets, and
a bottom plate that is adhered to the top plate; FIG. 7c depicts an
enlarged cross-section of a gasket molded into a pocket of the top plate;
FIG. 7D depicts a sideways perspective of the microfluidic chip depicted
in FIG. 7A.

[0026]FIG. 8A depicts a front/top perspective of an exemplary embodiment
of a gasket interface for use with a microfluidic chip according to the
invention; FIG. 8B depicts a side perspective of the gasket depicted in
FIG. 8A; FIG. 8C depicts a cross-section of the gasket interface depicted
in FIG. 8A.

[0027]FIG. 9A depicts a cross-section of the fluid interface with an
exemplary microfluidic chip using the gasket shown in FIG. 8A; FIG. 9B is
an enlarged perspective of the fluid interface shown in FIG. 9A.

[0028]FIG. 10A depicts a front/top perspective of an exemplary embodiment
of a gasket/chip/carrier assembly according to the invention; FIG. 10B
depicts a cross-section of a portion of the gasket/chip/carrier assembly
depicted in FIG. 10A; FIG. 10c depicts an enlarged perspective of a
portion of the gasket/chip/carrier shown in FIG. 10B.

[0029]FIG. 11A depicts a front/top perspective of an exemplary embodiment
of a gasket/chip/carrier assembly according to the invention; FIG. 11B
depicts a back/bottom perspective of the gasket/chip/carrier assembly
shown in FIG. 11A; FIG. 11c depicts an enlarged perspective of a portion
of the gasket/chip/carrier shown in FIG. 11B.

[0030]FIG. 12A depicts a front/top perspective of an exemplary embodiment
of a gasket/chip/carrier assembly according to the invention; FIG. 12B
depicts a back/bottom perspective of the gasket/chip/carrier shown in
FIG. 12B.

[0031] FIGS. 13A and 13B depict front perspectives of exemplary
embodiments of a gasket/chip/carrier assembly according to the invention.

[0032]FIG. 14A depicts a front/top perspective of an exemplary embodiment
of a gasket/chip/carrier assembly according to the invention; FIGS. 14B
and 14C depict different cross-sections of the gasket/chip/carrier
assembly shown in FIG. 14A; FIG. 14D depicts an enlarged detail of a
portion of FIG. 14B; FIG. 14E depicts an enlarged detail of a portion of
FIG. 14c.

[0033] FIG. 15A depicts a front/top perspective of an exemplary embodiment
of a gasket/chip/carrier assembly according to the invention; FIG. 15B
depicts an enlarged detail of a portion of FIG. 15A.

[0034]FIG. 16A depicts a schematic of an exemplary embodiment of a
gasket/chip/carrier assembly according to the invention; FIGS. 16B-16E
depict enlarged details of portions of FIG. 16A.

[0037]FIG. 19 illustrates the interconnects needed for each tube molded
into a single monolithic self-aligned part.

[0038]FIG. 20 shows a schematic of a molding tool based on this concept.
The pins are captured within an elastomeric molded sleeve and a
compression plate made from a rigid backer plate and foam rubber is used
to apply gentle even pressure to the pins and generate the force needed
to make the pins uniformly contact the master.

DETAILED DESCRIPTION

[0039] The microfluidic devices and methods of use described herein are
based on the creation and manipulation of aqueous phase droplets
completely encapsulated by an inert immiscible oil stream. This
combination enables precise droplet generation, highly efficient,
electrically addressable, droplet coalescence, and controllable,
electrically addressable single droplet sorting. The microfluidic devices
include one or more channels and modules. The integration of these
modules is an essential enabling technology for a droplet based,
high-throughput microfluidic reactor system.

[0040] The microfluidic devices of the present invention can be utilized
for numerous biological, chemical, or diagnostic applications, as
described in further detail herein.

Substrates

[0041] The microfluidic device of the present invention includes one or
more analysis units. An "analysis unit" is a micro substrate, e.g., a
microchip. The terms microsubstrate, substrate, microchip, and chip are
used interchangeably herein. The analysis unit includes at least one
inlet channel, at least one main channel, at least one inlet module, at
least one coalescence module, and at least one detection module. The
analysis unit can further include one or more sorting modules. The
sorting module can be in fluid communication with branch channels which
are in fluid communication with one or more outlet modules (collection
module or waste module). For sorting applications, at least one detection
module cooperates with at least one sorting module to divert flow via a
detector-originated signal. It shall be appreciated that the "modules"
and "channels" are in fluid communication with each other and therefore
may overlap; i.e., there may be no clear boundary where a module or
channel begins or ends. A plurality of analysis units of the invention
may be combined in one device. The analysis unit and specific modules are
described in further detail herein.

[0042] The dimensions of the substrate are those of typical microchips,
ranging between about 0.5 cm to about 15 cm per side and about 1 micron
to about 1 cm in thickness. A substrate can be transparent and can be
covered with a material having transparent properties, such as a glass
coverslip, to permit detection of a reporter, for example, by an optical
device such as an optical microscope. The material can be perforated for
functional interconnects, such as fluidic, electrical, and/or optical
interconnects, and sealed to the back interface of the device so that the
junction of the interconnects to the device is leak-proof. Such a device
can allow for application of high pressure to fluid channels without
leaking.

[0043] A variety of materials and methods, according to certain aspects of
the invention, can be used to form any of the described components of the
systems and devices of the invention. In some cases, the various
materials selected lend themselves to various methods. For example,
various components of the invention can be formed from solid materials,
in which the channels can be formed via molding, micromachining, film
deposition processes such as spin coating and chemical vapor deposition,
laser fabrication, photolithographic techniques, etching methods
including wet chemical or plasma processes, and the like. See, for
example, Scientific American, 248:44-55, 1983 (Angell, et al). At least a
portion of the fluidic system can be formed of silicone by molding a
silicone chip. Technologies for precise and efficient formation of
various fluidic systems and devices of the invention from silicone are
known. Various components of the systems and devices of the invention can
also be formed of a polymer, for example, an elastomeric polymer such as
polydimethylsiloxane ("PDMS"), polytetrafluoroethylene ("PTFE") or
Teflon® or the like.

[0044] The channels of the invention can be formed, for example by etching
a silicon chip using conventional photolithography techniques, or using a
micromachining technology called "soft lithography" as described by
Whitesides and Xia, Angewandte Chemie International Edition 37, 550
(1998). These and other methods may be used to provide inexpensive
miniaturized devices, and in the case of soft lithography, can provide
robust devices having beneficial properties such as improved flexibility,
stability, and mechanical strength. When optical detection is employed,
the invention also provides minimal light scatter from molecule, cell,
small molecule or particle suspension and chamber material.

[0045] Different components can be formed of different materials. For
example, a base portion including a bottom wall and side walls can be
formed from an opaque material such as silicone or PDMS, and a top
portion can be formed from a transparent or at least partially
transparent material, such as glass or a transparent polymer, for
observation and/or control of the fluidic process. Components can be
coated so as to expose a desired chemical functionality to fluids that
contact interior channel walls, where the base supporting material does
not have a precise, desired functionality. For example, components can be
formed as illustrated, with interior channel walls coated with another
material. Material used to form various components of the systems and
devices of the invention, e.g., materials used to coat interior walls of
fluid channels, may desirably be selected from among those materials that
will not adversely affect or be affected by fluid flowing through the
fluidic system, e.g., material(s) that is chemically inert in the
presence of fluids to be used within the device.

[0046] Various components of the invention when formed from polymeric
and/or flexible and/or elastomeric materials, and can be conveniently
formed of a hardenable fluid, facilitating formation via molding (e.g.
replica molding, injection molding, cast molding, etc.). The hardenable
fluid can be essentially any fluid that can be induced to solidify, or
that spontaneously solidifies, into a solid capable of containing and/or
transporting fluids contemplated for use in and with the fluidic network.
In one embodiment, the hardenable fluid comprises a polymeric liquid or a
liquid polymeric precursor (i.e. a "prepolymer"). Suitable polymeric
liquids can include, for example, thermoplastic polymers, thermoset
polymers, or mixture of such polymers heated above their melting point.
As another example, a suitable polymeric liquid may include a solution of
one or more polymers in a suitable solvent, which solution forms a solid
polymeric material upon removal of the solvent, for example, by
evaporation. Such polymeric materials, which can be solidified from, for
example, a melt state or by solvent evaporation, are well known to those
of ordinary skill in the art. A variety of polymeric materials, many of
which are elastomeric, are suitable, and are also suitable for forming
molds or mold masters, for embodiments where one or both of the mold
masters is composed of an elastomeric material. A non-limiting list of
examples of such polymers includes polymers of the general classes of
silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers
are characterized by the presence of a three-membered cyclic ether group
commonly referred to as an epoxy group, 1,2-epoxide, or oxirane. For
example, diglycidyl ethers of bisphenol A can be used, in addition to
compounds based on aromatic amine, triazine, and cycloaliphatic
backbones. Another example includes the well-known Novolac polymers.
Non-limiting examples of silicone elastomers suitable for use according
to the invention include those formed from precursors including the
chlorosilanes such as methylchlorosilanes, ethylchlorosilanes,
phenylchlorosilanes, etc.

[0047] Silicone polymers are preferred, for example, the silicone
elastomer polydimethylsiloxane. Non-limiting examples of PDMS polymers
include those sold under the trademark Sylgard by Dow Chemical Co.,
Midland, Mich., such as Sylgard 182, Sylgard 184, and Sylgard 186.
Silicone polymers including PDMS have several beneficial properties
simplifying formation of the microfluidic structures of the invention.
For instance, such materials are inexpensive, readily available, and can
be solidified from a prepolymeric liquid via curing with heat. For
example, PDMSs are typically curable by exposure of the prepolymeric
liquid to temperatures of about, for example, about 65° C. to
about 75° C. for exposure times of, for example, about an hour.
Also, silicone polymers, such as PDMS, can be elastomeric and thus may be
useful for forming very small features with relatively high aspect
ratios, necessary in certain embodiments of the invention. Flexible
(e.g., elastomeric) molds or masters can be advantageous in this regard.

[0048] The present invention provides improved methods of bonding PDMS to
incompatible media. Normal methods of bonding various materials (plastic,
metals, etc.) directly to materials such as PDMS, silicone, Teflon, and
PEEK using traditional bonding practices (adhesives, epoxies, etc.) do
not work well due to the poor adhesion of the bonding agent to materials
such as PDMS. Normal surface preparation by commercially available
surface activators has not worked well in microfluidic device
manufacturing. This problem is eliminated by treating the PDMS surface to
be bonded with high intensity oxygen or air plasma. The process converts
the top layer of PDMS to glass which bonds extremely well with normal
adhesives. Tests using this method to bond external fluid lines to PDMS
using a UV-cure adhesive (Loctite 352, 363, and others) resulted in a
bond that is stronger than the PDMS substrate, resulting in fracture of
the PDMS prior to failure of the bond. The present method combines high
radiant flux, wavelength selection, and cure exposure time to
significantly enhance the bond strength of the adhesive.

[0049] One advantage of forming structures such as microfluidic structures
of the invention from silicone polymers, such as PDMS, is the ability of
such polymers to be oxidized, for example by exposure to an
oxygen-containing plasma such as an air plasma, so that the oxidized
structures contain, at their surface, chemical groups capable of
cross-linking to other oxidized silicone polymer surfaces or to the
oxidized surfaces of a variety of other polymeric and non-polymeric
materials. Thus, components can be formed and then oxidized and
essentially irreversibly sealed to other silicone polymer surfaces, or to
the surfaces of other substrates reactive with the oxidized silicone
polymer surfaces, without the need for separate adhesives or other
sealing means. In most cases, sealing can be completed simply by
contacting an oxidized silicone surface to another surface without the
need to apply auxiliary pressure to form the seal. That is, the
pre-oxidized silicone surface acts as a contact adhesive against suitable
mating surfaces. Specifically, in addition to being irreversibly sealable
to itself, oxidized silicone such as oxidized PDMS can also be sealed
irreversibly to a range of oxidized materials other than itself
including, for example, glass, silicon, silicon oxide, quartz, silicon
nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers,
which have been oxidized in a similar fashion to the PDMS surface (for
example, via exposure to an oxygen-containing plasma). Oxidation and
sealing methods useful in the context of the present invention, as well
as overall molding techniques, are described in the art, for example, in
an article entitled "Rapid Prototyping of Microfluidic Systems and
Polydimethylsiloxane," Anal. Chem., 70:474-480, 1998 (Duffy et al.),
incorporated herein by reference.

[0050] Another advantage to forming microfluidic structures of the
invention (or interior, fluid-contacting surfaces) from oxidized silicone
polymers is that these surfaces can be much more hydrophilic than the
surfaces of typical elastomeric polymers (where a hydrophilic interior
surface is desired). Such hydrophilic channel surfaces can thus be more
easily filled and wetted with aqueous solutions than can structures
comprised of typical, unoxidized elastomeric polymers or other
hydrophobic materials.

[0051] In one embodiment, a bottom wall is formed of a material different
from one or more side walls or a top wall, or other components. For
example, the interior surface of a bottom wall can comprise the surface
of a silicon wafer or microchip, or other substrate. Other components
can, as described above, be sealed to such alternative substrates. Where
it is desired to seal a component comprising a silicone polymer (e.g.
PDMS) to a substrate (bottom wall) of different material, the substrate
may be selected from the group of materials to which oxidized silicone
polymer is able to irreversibly seal (e.g., glass, silicon, silicon
oxide, quartz, silicon nitride, polyethylene, polystyrene, epoxy
polymers, and glassy carbon surfaces which have been oxidized).
Alternatively, other sealing techniques can be used, as would be apparent
to those of ordinary skill in the art, including, but not limited to, the
use of separate adhesives, thermal bonding, solvent bonding, ultrasonic
welding, etc.

Channels

[0052] The microfluidic substrates of the present invention include
channels that form the boundary for a fluid. A "channel," as used herein,
means a feature on or in a substrate that at least partially directs the
flow of a fluid. In some cases, the channel may be formed, at least in
part, by a single component, e.g., an etched substrate or molded unit.
The channel can have any cross-sectional shape, for example, circular,
oval, triangular, irregular, square or rectangular (having any aspect
ratio), or the like, and can be covered or uncovered (i.e., open to the
external environment surrounding the channel). In embodiments where the
channel is completely covered, at least one portion of the channel can
have a cross-section that is completely enclosed, and/or the entire
channel may be completely enclosed along its entire length with the
exception of its inlet and outlet.

[0053] An open channel generally will include characteristics that
facilitate control over fluid transport, e.g., structural characteristics
(an elongated indentation) and/or physical or chemical characteristics
(hydrophobicity vs. hydrophilicity) and/or other characteristics that can
exert a force (e.g., a containing force) on a fluid. The fluid within the
channel may partially or completely fill the channel. In some cases the
fluid may be held or confined within the channel or a portion of the
channel in some fashion, for example, using surface tension (e.g., such
that the fluid is held within the channel within a meniscus, such as a
concave or convex meniscus). In an article or substrate, some (or all) of
the channels may be of a particular size or less, for example, having a
largest dimension perpendicular to fluid flow of less than about 5 mm,
less than about 2 mm, less than about 1 mm, less than about 500 microns,
less than about 200 microns, less than about 100 microns, less than about
60 microns, less than about 50 microns, less than about 40 microns, less
than about 30 microns, less than about 25 microns, less than about 10
microns, less than about 3 microns, less than about 1 micron, less than
about 300 nm, less than about 100 nm, less than about 30 nm, or less than
about 10 nm or less in some cases. Of course, in some cases, larger
channels, tubes, etc. can be used to store fluids in bulk and/or deliver
a fluid to the channel. In one embodiment, the channel is a capillary.

[0054] The dimensions of the channel may be chosen such that fluid is able
to freely flow through the channel, for example, if the fluid contains
cells. The dimensions of the channel may also be chosen, for example, to
allow a certain volumetric or linear flow rate of fluid in the channel.
Of course, the number of channels and the shape of the channels can be
varied by any method known to those of ordinary skill in the art. In some
cases, more than one channel or capillary may be used. For example, two
or more channels may be used, where they are positioned inside each
other, positioned adjacent to each other, etc.

[0055] For particles (e.g., cells) or molecules that are in droplets
(i.e., deposited by the inlet module) within the flow of the main
channel, the channels of the device are preferably square, with a
diameter between about 2 microns and 1 mm. This geometry facilitates an
orderly flow of droplets in the channels. Similarly, the volume of the
detection module in an analysis device is typically in the range of
between about 0.1 picoliters and 500 nanoliters.

[0056] A "main channel" is a channel of the device of the invention which
permits the flow of molecules, cells, small molecules or particles past a
coalescence module for coalescing one or more droplets, a detection
module for detection (identification) or measurement of a droplet and a
sorting module, if present, for sorting a droplet based on the detection
in the detection module. The main channel is typically in fluid
communication with the coalescence, detection and/or sorting modules, as
well as, an inlet channel of the inlet module. The main channel is also
typically in fluid communication with an outlet module and optionally
with branch channels, each of which may have a collection module or waste
module. These channels permit the flow of molecules, cells, small
molecules or particles out of the main channel. An "inlet channel"
permits the flow of molecules, cells, small molecules or particles into
the main channel. One or more inlet channels communicate with one or more
means for introducing a sample into the device of the present invention.
The inlet channel communicates with the main channel at an inlet module.

[0057] The microfluidic substrate can also comprise one or more fluid
channels to inject or remove fluid in between droplets in a droplet
stream for the purpose of changing the spacing between droplets.

[0058] The channels of the device of the present invention can be of any
geometry as described. However, the channels of the device can comprise a
specific geometry such that the contents of the channel are manipulated,
e.g., sorted, mixed, prevent clogging, etc.

[0059] A microfluidic substrate can also include a specific geometry
designed in such a manner as to prevent the aggregation of
biological/chemical material and keep the biological/chemical material
separated from each other prior to encapsulation in droplets. The
geometry of channel dimension can be changed to disturb the aggregates
and break them apart by various methods, that can include, but is not
limited to, geometric pinching (to force cells through a (or a series of)
narrow region(s), whose dimension is smaller or comparable to the
dimension of a single cell) or a barricade (place a series of barricades
on the way of the moving cells to disturb the movement and break up the
aggregates of cells).

[0060] To prevent material (e.g., cells and other particles or molecules)
from adhering to the sides of the channels, the channels (and coverslip,
if used) may have a coating which minimizes adhesion. Such a coating may
be intrinsic to the material from which the device is manufactured, or it
may be applied after the structural aspects of the channels have been
microfabricated. "TEFLON" is an example of a coating that has suitable
surface properties. The surface of the channels of the microfluidic
device can be coated with any anti-wetting or blocking agent for the
dispersed phase. The channel can be coated with any protein to prevent
adhesion of the biological/chemical sample. For example, in one
embodiment the channels are coated with BSA, PEG-silane and/or
fluorosilane. For example, 5 mg/ml BSA is sufficient to prevent
attachment and prevent clogging. In another embodiment, the channels can
be coated with a cyclized transparent optical polymer obtained by
copolymerization of perfluoro (alkenyl vinyl ethers), such as the type
sold by Asahi Glass Co. under the trademark Cytop. In such an embodiment,
the coating is applied from a 0.1-0.5 wt % solution of Cytop CTL-809M in
CT-Solv 180. This solution can be injected into the channels of a
microfluidic device via a plastic syringe. The device can then be heated
to about 90° C. for 2 hours, followed by heating at 200° C.
for an additional 2 hours. In another embodiment, the channels can be
coated with a hydrophobic coating of the type sold by PPG Industries,
Inc. under the trademark Aquapel (e.g., perfluoroalkylalkylsilane surface
treatment of plastic and coated plastic substrate surfaces in conjunction
with the use of a silica primer layer) and disclosed in U.S. Pat. No.
5,523,162, which patent is hereby incorporated by reference in its
entirety. By fluorinating the surfaces of the channels, the continuous
phase preferentially wets the channels and allows for the stable
generation and movement of droplets through the device. The low surface
tension of the channel walls thereby minimizes the accumulation of
channel clogging particulates.

[0061] The surface of the channels in the microfluidic device can be also
fluorinated to prevent undesired wetting behaviors. For example, a
microfluidic device can be placed in a polycarbonate dessicator with an
open bottle of (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane.
The dessicator is evacuated for 5 minutes, and then sealed for 2040
minutes. The dessicator is then backfilled with air and removed. This
approach uses a simple diffusion mechanism to enable facile infiltration
of channels of the microfluidic device with the fluorosilane and can be
readily scaled up for simultaneous device fluorination.

Fluids

[0062] The microfluidic device of the present invention is capable of
controlling the direction and flow of fluids and entities within the
device. The term "flow" means any movement of liquid or solid through a
device or in a method of the invention, and encompasses without
limitation any fluid stream, and any material moving with, within or
against the stream, whether or not the material is carried by the stream.
For example, the movement of molecules, beads, cells or virions through a
device or in a method of the invention, e.g. through channels of a
microfluidic chip of the invention, comprises a flow. This is so,
according to the invention, whether or not the molecules, beads, cells or
virions are carried by a stream of fluid also comprising a flow, or
whether the molecules, cells or virions are caused to move by some other
direct or indirect force or motivation, and whether or not the nature of
any motivating force is known or understood. The application of any force
may be used to provide a flow, including without limitation, pressure,
capillary action, electro-osmosis, electrophoresis, dielectrophoresis,
optical tweezers, and combinations thereof, without regard for any
particular theory or mechanism of action, so long as molecules, cells or
virions are directed for detection, measurement or sorting according to
the invention. Specific flow forces are described in further detail
herein.

[0063] The flow stream in the main channel is typically, but not
necessarily, continuous and may be stopped and started, reversed or
changed in speed. A liquid that does not contain sample molecules, cells
or particles can be introduced into a sample inlet well or channel and
directed through the inlet module, e.g., by capillary action, to hydrate
and prepare the device for use. Likewise, buffer or oil can also be
introduced into a main inlet region that communicates directly with the
main channel to purge the device (e.g., or "dead" air) and prepare it for
use. If desired, the pressure can be adjusted or equalized, for example,
by adding buffer or oil to an outlet module.

[0064] As used herein, the term "fluid stream" or "fluidic stream" refers
to the flow of a fluid, typically generally in a specific direction. The
fluidic stream may be continuous and/or discontinuous. A "continuous"
fluidic stream is a fluidic stream that is produced as a single entity,
e.g., if a continuous fluidic stream is produced from a channel, the
fluidic stream, after production, appears to be contiguous with the
channel outlet. The continuous fluidic stream is also referred to as a
continuous phase fluid or carrier fluid. The continuous fluidic stream
may be laminar, or turbulent in some cases.

[0065] Similarly, a "discontinuous" fluidic stream is a fluidic stream
that is not produced as a single entity. The discontinuous fluidic stream
is also referred to as the dispersed phase fluid or sample fluid. A
discontinuous fluidic stream may have the appearance of individual
droplets, optionally surrounded by a second fluid. A "droplet," as used
herein, is an isolated portion of a first fluid that completely
surrounded by a second fluid. In some cases, the droplets may be
spherical or substantially spherical; however, in other cases, the
droplets may be non-spherical, for example, the droplets may have the
appearance of "blobs" or other irregular shapes, for instance, depending
on the external environment. As used herein, a first entity is
"surrounded" by a second entity if a closed loop can be drawn or
idealized around the first entity through only the second entity. The
dispersed phase fluid can include a biological/chemical material. The
biological/chemical material can be tissues, cells, particles, proteins,
antibodies, amino acids, nucleotides, small molecules, and
pharmaceuticals. The biological/chemical material can include one or more
labels known in the art. The label can be a DNA tag, dyes or quantum dot,
or combinations thereof.

Droplets

[0066] The term "emulsion" refers to a preparation of one liquid
distributed in small globules (also referred to herein as drops, droplets
or NanoReactors) in the body of a second liquid. The first and second
fluids are immiscible with each other. For example, the discontinuous
phase can be an aqueous solution and the continuous phase can be a
hydrophobic fluid such as an oil. This is termed a water-in-oil emulsion.
Alternatively, the emulsion may be an oil-in-water emulsion. In that
example, the first liquid, which is dispersed in globules, is referred to
as the discontinuous phase, whereas the second liquid is referred to as
the continuous phase or the dispersion medium. The continuous phase can
be an aqueous solution and the discontinuous phase is a hydrophobic
fluid, such as an oil (e.g., decane, tetradecane, or hexadecane). The
droplets or globules of oil in an oil-in-water emulsion are also referred
to herein as "micelles", whereas globules of water in a water-in-oil
emulsion may be referred to as "reverse micelles".

[0067] The fluidic droplets may each be substantially the same shape
and/or size. The shape and/or size can be determined, for example, by
measuring the average diameter or other characteristic dimension of the
droplets. The "average diameter" of a plurality or series of droplets is
the arithmetic average of the average diameters of each of the droplets.
Those of ordinary skill in the art will be able to determine the average
diameter (or other characteristic dimension) of a plurality or series of
droplets, for example, using laser light scattering, microscopic
examination, or other known techniques. The diameter of a droplet, in a
non-spherical droplet, is the mathematically-defined average diameter of
the droplet, integrated across the entire surface. The average diameter
of a droplet (and/or of a plurality or series of droplets) may be, for
example, less than about 1 mm, less than about 500 micrometers, less than
about 200 micrometers, less than about 100 micrometers, less than about
75 micrometers, less than about 50 micrometers, less than about 25
micrometers, less than about 10 micrometers, or less than about 5
micrometers in some cases. The average diameter may also be at least
about 1 micrometer, at least about 2 micrometers, at least about 3
micrometers, at least about 5 micrometers, at least about 10 micrometers,
at least about 15 micrometers, or at least about 20 micrometers in
certain cases.

[0068] As used herein, the term "NanoReactor" and its plural encompass the
terms "droplet", "nanodrop", "nanodroplet", "microdrop" or "microdroplet"
as defined herein, as well as an integrated system for the manipulation
and probing of droplets, as described in detail herein. Nanoreactors as
described herein can be 0.1-1000 μm (e.g., 0.1, 0.2 . . . 5, 10, 15,
20, 25, 30, 35, 40, 45, 50 . . . 1000 μm), or any size within in this
range. Droplets at these dimensions tend to conform to the size and shape
of the channels, while maintaining their respective volumes. Thus, as
droplets move from a wider channel to a narrower channel they become
longer and thinner, and vice versa.

[0069] The microfluidic substrate of this invention most preferably
generates round, monodisperse droplets. The droplets can have a diameter
that is smaller than the diameter of the microchannel; i.e., preferably
15 to 100 μm when cells are used; or 10 to 75 μm when reagents or
other chemical or biological agents are used; or 100 to 1000 μm when
droplets are used for sequencing reactions such that droplets will be
removed and dispensed into other collection apparatuses, such as
microtiter plates or utilized in sequencing devices. Monodisperse
droplets are particularly preferably, e.g., in high throughput devices
and other embodiments where it is desirable to generate droplets at high
frequency and of high uniformity.

[0070] The droplet forming liquid is typically an aqueous buffer solution,
such as ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for
example by column chromatography), 10 mM Tris HCl and 1 mM EDTA (TE)
buffer, phosphate buffer saline (PBS) or acetate buffer. Any liquid or
buffer that is physiologically compatible with the population of
molecules, cells or particles to be analyzed and/or sorted can be used.
The fluid passing through the main channel and in which the droplets are
formed is one that is immiscible with the droplet forming fluid. The
fluid passing through the main channel can be a non-polar solvent, decane
(e.g., tetradecane or hexadecane), fluorocarbon oil, silicone oil or
another oil (for example, mineral oil).

[0071] The dispersed phase fluid may also contain biological/chemical
material (e.g., molecules, cells, or other particles) for combination,
analysis and/or sorting in the device. The droplets of the dispersed
phase fluid can contain more than one particle or can contain no more
than one particle. For example, where the biological material comprises
cells, each droplet preferably contains, on average, no more than one
cell. However, in some embodiments, each droplet may contain, on average,
at least 1000 cells. The droplets can be detected and/or sorted according
to their contents.

[0072] The concentration (i.e., number) of molecules, cells or particles
in a droplet can influence sorting efficiently and therefore is
preferably optimized. In particular, the sample concentration should be
dilute enough that most of the droplets contain no more than a single
molecule, cell or particle, with only a small statistical chance that a
droplet will contain two or more molecules, cells or particles. This is
to ensure that for the large majority of measurements, the level of
reporter measured in each droplet as it passes through the detection
module corresponds to a single molecule, cell or particle and not to two
or more molecules, cells or particles.

[0073] The parameters which govern this relationship are the volume of the
droplets and the concentration of molecules, cells or particles in the
sample solution. The probability that a droplet will contain two or more
molecules, cells or particles (P≦2) can be expressed as

P≦2=1-{1+[cell]×v}×e-[cell]×V

[0074] where "[cell]" is the concentration of molecules, cells or
particles in units of number of molecules, cells or particles per cubic
micron (μm3), and V is the volume of the droplet in units of
μm3.

[0075] It will be appreciated that P≦2 can be minimized by
decreasing the concentration of molecules, cells or particles in the
sample solution. However, decreasing the concentration of molecules,
cells or particles in the sample solution also results in an increased
volume of solution processed through the device and can result in longer
run times. Accordingly, it is desirable to minimize to presence of
multiple molecules, cells or particles in the droplets (thereby
increasing the accuracy of the sorting) and to reduce the volume of
sample, thereby permitting a sorted sample in a reasonable time in a
reasonable volume containing an acceptable concentration of molecules,
cells or particles.

[0076] The maximum tolerable P≦2 depends on the desired
"purity" of the sorted sample. The "purity" in this case refers to the
fraction of sorted molecules, cells or particles that possess a desired
characteristic (e.g., display a particular antigen, are in a specified
size range or are a particular type of molecule, cell or particle). The
purity of the sorted sample is inversely proportional to P≦2.
For example, in applications where high purity is not needed or desired a
relatively high P≦2 (e.g., P≦2=0.2) may be
acceptable. For most applications, maintaining P≦2 at or
below about 0.1, preferably at or below about 0.01, provides satisfactory
results.

[0077] The fluids used to generate droplets in microfluidic devices are
typically immiscible liquids such as oil and water. These two materials
generally have very different dielectric constants associated with them.
These differences can be exploited to determine droplet rate and size for
every drop passing through a small section of a microfluidic device. One
method to directly monitor this variation in the dielectric constant
measures the change in capacitance over time between a pair of closely
spaced electrodes. This change in capacitance can be detected by the
change in current measured in these electrodes:

i=V×dC/dt

[0078] Where i is the current, V is the voltage applied across the
electrodes, and dC/dt is the change in capacitance with time.
Alternatively, the capacitance can be measured directly if a time varying
voltage is applied to these same electrodes: i=C×dV/dt Where C is
the measured capacitance, and dV/dt is the change in voltage with time.
As a first approximation, the electrode pair can be determined as a
parallel plate capacitor:

C=.di-elect cons.0k×A/d

[0079] Where .di-elect cons.0 is the permittivity of free space, k is
the effective dielectric constant (this changes every time a droplet
passes through), A is the area of the capacitor and d is the electrode
separation. The current measured in the device is then plotted as a
function of time.

[0080] The fluidic droplets may contain additional entities, for example,
other chemical, biochemical, or biological entities (e.g., dissolved or
suspended in the fluid), cells, particles, gases, molecules, or the like.
In some cases, the droplets may each be substantially the same shape or
size, as discussed above. In certain instances, the invention provides
for the production of droplets consisting essentially of a substantially
uniform number of entities of a species therein (i.e., molecules, cells,
particles, etc.). For example, about 90%, about 93%, about 95%, about
97%, about 98%, or about 99%, or more of a plurality or series of
droplets may each contain the same number of entities of a particular
species. For instance, a substantial number of fluidic droplets produced,
e.g., as described above, may each contain 1 entity, 2 entities, 3
entities, 4 entities, 5 entities, 7 entities, 10 entities, 15 entities,
20 entities, 25 entities, 30 entities, 40 entities, 50 entities, 60
entities, 70 entities, 80 entities, 90 entities, 100 entities, etc.,
where the entities are molecules or macromolecules, cells, particles,
etc. In some cases, the droplets may each independently contain a range
of entities, for example, less than 20 entities, less than 15 entities,
less than 10 entities, less than 7 entities, less than 5 entities, or
less than 3 entities in some cases. In some embodiments, a droplet may
contain 100,000,000 entities. In other embodiments, a droplet may contain
1,000,000 entities.

[0081] In a liquid containing droplets of fluid, some of which contain a
species of interest and some of which do not contain the species of
interest, the droplets of fluid may be screened or sorted for those
droplets of fluid containing the species as further described below
(e.g., using fluorescence or other techniques such as those described
above), and in some cases, the droplets may be screened or sorted for
those droplets of fluid containing a particular number or range of
entities of the species of interest, e.g., as previously described. Thus,
in some cases, a plurality or series of fluidic droplets, some of which
contain the species and some of which do not, may be enriched (or
depleted) in the ratio of droplets that do contain the species, for
example, by a factor of at least about 2, at least about 3, at least
about 5, at least about 10, at least about 15, at least about 20, at
least about 50, at least about 100, at least about 125, at least about
150, at least about 200, at least about 250, at least about 500, at least
about 750, at least about 1000, at least about 2000, or at least about
5000 or more in some cases. In other cases, the enrichment (or depletion)
may be in a ratio of at least about 104, at least about 105, at
least about 106, at least about 107, at least about 108,
at least about 109, at least about 1010, at least about
1011, at least about 1012, at least about 1013, at least
about 1014, at least about 1015, or more. For example, a
fluidic droplet containing a particular species may be selected from a
library of fluidic droplets containing various species, where the library
may have about 100, about 103, about 104, about 105, about
106, about 107, about 108, about 109, about
1010, about 1011, about 1012, about 1013, about
1014, about 1015, or more items, for example, a DNA library, an
RNA library, a protein library, a combinatorial chemistry library, etc.
In certain embodiments, the droplets carrying the species may then be
fused, reacted, or otherwise used or processed, etc., as further
described below, for example, to initiate or determine a reaction.

[0082] Droplets of a sample fluid can be formed within the inlet module on
the microfluidic device or droplets (or droplet libraries) can be formed
before the sample fluid is introduced to the microfluidic device ("off
chip" droplet formation). To permit effective interdigitation,
coalescence and detection, the droplets comprising each sample to be
analyzed must be monodisperse. As described in more detail herein, in
many applications, different samples to be analyzed are contained within
droplets of different sizes. Droplet size must be highly controlled to
ensure that droplets containing the correct contents for analysis and
coalesced properly. As such, the present invention provides devices and
methods for forming droplets and droplet libraries.

Surfactants

[0083] The fluids used in the invention may contain one or more additives,
such as agents which reduce surface tensions (surfactants). Surfactants
can include Tween, Span, fluorosurfactants, and other agents that are
soluble in oil relative to water. In some applications, performance is
improved by adding a second surfactant to the aqueous phase. Surfactants
can aid in controlling or optimizing droplet size, flow and uniformity,
for example by reducing the shear force needed to extrude or inject
droplets into an intersecting channel. This can affect droplet volume and
periodicity, or the rate or frequency at which droplets break off into an
intersecting channel. Furthermore, the surfactant can serve to stabilize
aqueous emulsions in fluorinated oils from coalescing.

[0084] The droplets may be coated with a surfactant. Preferred surfactants
that may be added to the continuous phase fluid include, but are not
limited to, surfactants such as sorbitan-based carboxylic acid esters
(e.g., the "Span" surfactants, Fluka Chemika), including sorbitan
monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan
monostearate (Span 60) and sorbitan monooleate (Span 80), and
perfluorinated polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/or FSH).
Other non-limiting examples of non-ionic surfactants which may be used
include polyoxyethylenated alkylphenols (for example, nonyl-, p-dodecyl-,
and dinonylphenols), polyoxyethylenated straight chain alcohols,
polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated
mercaptans, long chain carboxylic acid esters (for example, glyceryl and
polyglycerl esters of natural fatty acids, propylene glycol, sorbitol,
polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters, etc.)
and alkanolamines (e.g., diethanolamine-fatty acid condensates and
isopropanolamine-fatty acid condensates). In addition, ionic surfactants
such as sodium dodecyl sulfate (SDS) may also be used. However, such
surfactants are generally less preferably for many embodiments of the
invention. For instance, in those embodiments where aqueous droplets are
used as nanoreactors for chemical reactions (including biochemical
reactions) or are used to analyze and/or sort biomaterials, a water
soluble surfactant such as SDS may denature or inactivate the contents of
the droplet.

[0085] The carrier fluid can be an oil (e.g., decane, tetradecane or
hexadecane) or fluorocarbon oil that contains a surfactant (e.g., a
non-ionic surfactant such as a Span surfactant) as an additive
(preferably between about 0.2 and 5% by volume, more preferably about
2%). A user can preferably cause the carrier fluid to flow through
channels of the microfluidic device so that the surfactant in the carrier
fluid coats the channel walls.

[0086] In one embodiment, the fluorosurfactant can be prepared by reacting
the perflourinated polyether DuPont Krytox 157 FSL, FSM, or FSH with
aqueous ammonium hydroxide in a volatile fluorinated solvent. The solvent
and residual water and ammonia can be removed with a rotary evaporator.
The surfactant can then be dissolved (e.g., 2.5 wt %) in a fluorinated
oil (e.g., Flourinert (3M)), which then serves as the continuous phase of
the emulsion.

Driving Forces

[0087] The invention can use pressure drive flow control, e.g., utilizing
valves and pumps, to manipulate the flow of cells, particles, molecules,
enzymes or reagents in one or more directions and/or into one or more
channels of a microfluidic device. However, other methods may also be
used, alone or in combination with pumps and valves, such as
electro-osmotic flow control, electrophoresis and dielectrophoresis
(Fulwyer, Science 156, 910 (1974); Li and Harrison, Analytical Chemistry
69, 1564 (1997); Fiedler, et al. Analytical Chemistry 70, 1909-1915
(1998); U.S. Pat. No. 5,656,155). Application of these techniques
according to the invention provides more rapid and accurate devices and
methods for analysis or sorting, for example, because the sorting occurs
at or in a sorting module that can be placed at or immediately after a
detection module. This provides a shorter distance for molecules or cells
to travel, they can move more rapidly and with less turbulence, and can
more readily be moved, examined, and sorted in single file, i.e., one at
a time.

[0088] Positive displacement pressure driven flow is a preferred way of
controlling fluid flow and dielectrophoresis is a preferred way of
manipulating droplets within that flow.

[0089] The pressure at the inlet module can also be regulated by adjusting
the pressure on the main and sample inlet channels, for example, with
pressurized syringes feeding into those inlet channels. By controlling
the pressure difference between the oil and water sources at the inlet
module, the size and periodicity of the droplets generated may be
regulated. Alternatively, a valve may be placed at or coincident to
either the inlet module or the sample inlet channel connected thereto to
control the flow of solution into the inlet module, thereby controlling
the size and periodicity of the droplets. Periodicity and droplet volume
may also depend on channel diameter, the viscosity of the fluids, and
shear pressure.

[0090] Without being bound by any theory, electro-osmosis is believed to
produce motion in a stream containing ions e.g. a liquid such as a
buffer, by application of a voltage differential or charge gradient
between two or more electrodes. Neutral (uncharged) molecules or cells
can be carried by the stream. Electro-osmosis is particularly suitable
for rapidly changing the course, direction or speed of flow.
Electrophoresis is believed to produce movement of charged objects in a
fluid toward one or more electrodes of opposite charge, and away from one
on or more electrodes of like charge. Where an aqueous phase is combined
with an oil phase, aqueous droplets are encapsulated or separated from
each other by oil. Typically, the oil phase is not an electrical
conductor and may insulate the droplets from the electro-osmotic field.
In this example, electro-osmosis may be used to drive the flow of
droplets if the oil is modified to carry or react to an electrical field,
or if the oil is substituted for another phase that is immiscible in
water but which does not insulate the water phase from electrical fields.

[0091] Dielectrophoresis is believed to produce movement of dielectric
objects, which have no net charge, but have regions that are positively
or negatively charged in relation to each other. Alternating,
non-homogeneous electric fields in the presence of droplets and/or
particles, such as cells or molecules, cause the droplets and/or
particles to become electrically polarized and thus to experience
dielectrophoretic forces. Depending on the dielectric polarizability of
the particles and the suspending medium, dielectric particles will move
either toward the regions of high field strength or low field strength.
For example, the polarizability of living cells depends on their
composition, morphology, and phenotype and is highly dependent on the
frequency of the applied electrical field. Thus, cells of different types
and in different physiological states generally possess distinctly
different dielectric properties, which may provide a basis for cell
separation, e.g., by differential dielectrophoretic forces. Likewise, the
polarizability of droplets also depends upon their size, shape and
composition. For example, droplets that contain salts can be polarized.
According to formulas provided in Fiedler, et al. Analytical Chemistry
70, 1909-1915 (1998), individual manipulation of single droplets requires
field differences (inhomogeneities) with dimensions close to the
droplets.

[0092] The term "dielectrophoretic force gradient" means a
dielectrophoretic force is exerted on an object in an electric field
provided that the object has a different dielectric constant than the
surrounding media. This force can either pull the object into the region
of larger field or push it out of the region of larger field. The force
is attractive or repulsive depending respectively on whether the object
or the surrounding media has the larger dielectric constant.

[0093] Manipulation is also dependent on permittivity (a dielectric
property) of the droplets and/or particles with the suspending medium.
Thus, polymer particles, living cells show negative dielectrophoresis at
high-field frequencies in water. For example, dielectrophoretic forces
experienced by a latex sphere in a 0.5 MV/m field (10 V for a 20 micron
electrode gap) in water are predicted to be about 0.2 piconewtons (pN)
for a 3.4 micron latex sphere to 15 pN for a 15 micron latex sphere
(Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998)). These values
are mostly greater than the hydrodynamic forces experienced by the sphere
in a stream (about 0.3 pN for a 3.4 micron sphere and 1.5 pN for a 15
micron sphere). Therefore, manipulation of individual cells or particles
can be accomplished in a streaming fluid, such as in a cell sorter
device, using dielectrophoresis. Using conventional semiconductor
technologies, electrodes can be microfabricated onto a substrate to
control the force fields in a microfabricated sorting device of the
invention. Dielectrophoresis is particularly suitable for moving objects
that are electrical conductors. The use of AC current is preferred, to
prevent permanent alignment of ions. Megahertz frequencies are suitable
to provide a net alignment, attractive force, and motion over relatively
long distances. See U.S. Pat. No. 5,454,472.

[0094] Radiation pressure can also be used in the invention to deflect and
move objects, e.g. droplets and particles (molecules, cells, particles,
etc.) contained therein, with focused beams of light such as lasers. Flow
can also be obtained and controlled by providing a pressure differential
or gradient between one or more channels of a device or in a method of
the invention.

[0095] Molecules, cells or particles (or droplets containing molecules,
cells or particles) can be moved by direct mechanical switching, e.g.,
with on-off valves or by squeezing the channels. Pressure control may
also be used, for example, by raising or lowering an output well to
change the pressure inside the channels on the chip. See, e.g., the
devices and methods described U.S. Pat. No. 6,540,895. These methods and
devices can further be used in combination with the methods and devices
described in pending U.S. Patent Application Publication No. 20010029983
and 20050226742. Different switching and flow control mechanisms can be
combined on one chip or in one device and can work independently or
together as desired.

Inlet Module

[0096] The microfluidic device of the present invention includes one or
more inlet modules. An "inlet module" is an area of a microfluidic
substrate device that receives molecules, cells, small molecules or
particles for additional coalescence, detection and/or sorting. The inlet
module can contain one or more inlet channels, wells or reservoirs,
openings, and other features which facilitate the entry of molecules,
cells, small molecules or particles into the substrate. A substrate may
contain more than one inlet module if desired. Different sample inlet
channels can communicate with the main channel at different inlet
modules. Alternately, different sample inlet channels can communication
with the main channel at the same inlet module. The inlet module is in
fluid communication with the main channel. The inlet module generally
comprises a junction between the sample inlet channel and the main
channel such that a solution of a sample (i.e., a fluid containing a
sample such as molecules, cells, small molecules (organic or inorganic)
or particles) is introduced to the main channel and forms a plurality of
droplets. The sample solution can be pressurized. The sample inlet
channel can intersect the main channel such that the sample solution is
introduced into the main channel at an angle perpendicular to a stream of
fluid passing through the main channel. For example, the sample inlet
channel and main channel intercept at a T-shaped junction; i.e., such
that the sample inlet channel is perpendicular (90 degrees) to the main
channel. However, the sample inlet channel can intercept the main channel
at any angle, and need not introduce the sample fluid to the main channel
at an angle that is perpendicular to that flow. The angle between
intersecting channels is in the range of from about 60 to about 120
degrees. Particular exemplary angles are 45, 60, 90, and 120 degrees.

[0097] Embodiments of the invention are also provided in which there are
two or more inlet modules introducing droplets of samples into the main
channel. For example, a first inlet module may introduce droplets of a
first sample into a flow of fluid in the main channel and a second inlet
module may introduce droplets of a second sample into the flow of fluid
in main channel, and so forth. The second inlet module is preferably
downstream from the first inlet module (e.g., about 30 μm). The fluids
introduced into the two or more different inlet modules can comprise the
same fluid or the same type of fluid (e.g., different aqueous solutions).
For example, droplets of an aqueous solution containing an enzyme are
introduced into the main channel at the first inlet module and droplets
of aqueous solution containing a substrate for the enzyme are introduced
into the main channel at the second inlet module. Alternatively, the
droplets introduced at the different inlet modules may be droplets of
different fluids which may be compatible or incompatible. For example,
the different droplets may be different aqueous solutions, or droplets
introduced at a first inlet module may be droplets of one fluid (e.g., an
aqueous solution) whereas droplets introduced at a second inlet module
may be another fluid (e.g., alcohol or oil).

Reservoir/Well

[0098] A device of the invention can include a sample solution reservoir
or well or other apparatus for introducing a sample to the device, at the
inlet module, which is typically in fluid communication with an inlet
channel. Reservoirs and wells used for loading one or more samples onto
the microfluidic device of the present invention, include but are not
limited to, syringes, pipettes, cartridges, vials, eppendorf tubes and
cell culture materials (e.g., 96 well plates). A reservoir may facilitate
introduction of molecules or cells into the device and into the sample
inlet channel of each analysis unit.

Fluidic Interconnects

[0099] The microfluidic device can include a pipette, a syringe (or other
glass container), or a tubing that is treated with a vapor or solution of
an appropriate PEG-silane to effect the surface PEG functionalization.
The purpose for treating the walls of glass containers (e.g., syringes)
with a PEG functionality is to prevent biological adhesion to the inner
walls of the container, which frustrates the proper transfer of
biological/chemical materials into the microfluidic device of the present
invention. The inlet channel is further connected to a means for
introducing a sample to said device. The means can be a well or
reservoir. The means can be temperature controlled. The inlet module may
also contain a connector adapted to receive a suitable piece of tubing,
such as liquid chromatography or HPLC tubing, through which a sample may
be supplied. Such an arrangement facilitates introducing the sample
solution under positive pressure in order to achieve a desired infusion
rate at the inlet module.

[0100] The interconnections, including tubes, must be extremely clean and
make excellent bonding with the PDMS surface in order to allow proper
operation of the device. The difficulty in making a fluidic connection to
a microfluidic device is primarily due to the difficulty in transitioning
from a macroscopic fluid line into the device while minimizing dead
volume.

[0101] In order to minimize contamination and leakage and allow for
greater reproducibility and reliability are improved, tubes and
interconnects for the PDMS slab can be cured in place. The tubes and
interconnects can be placed in position by applying a UV-cured adhesive
to allow for holding the tubes in place on the silicone wafer. Once the
tubes are placed in position, PDMS can be poured over the wafer and
cured. The cured PDMS, along with the tubes in place, can be peeled off
of the silicone wafer easily. This process can be applied to fluidics
channels as well as other connection channels. Once the adhesive is
applied onto the wafer, the process will allow for quick templating of
PDMS slabs with exact reproducibility of channel locations and
cleanliness. Tubes of any size can be implemented for this process. This
process allows for less stress on the interconnection joints and smaller
interconnection footprints in the device (see, for example,
PCT/US2006/02186 filed on Jun. 1, 2006; PCT/US2006/021280 filed on Jun.
1, 2006 and PCT/US2006/021380 filed on Jun. 1, 2006, each of which is
incorporated by reference in their entirety for all purposes).

[0102] The tubing side of the interconnect can be mounted into a retaining
block that provides precise registration of the tubing, while the
microfluidic device can be positioned accurately in a carrier that the
retaining block would align and clamp to. The total dead volume
associated with these designs would be critically dependent on how
accurately the two mating surfaces could be positioned relative to each
other. The maximum force required to maintain the seal would be limited
by the exact shape and composition of the sealing materials as well as
the rigidity and strength of the device itself. The shapes of the mating
surfaces can be tailored to the minimal leakage potential, sealing force
required, and potential for misalignment. By way of non-limiting example,
the single ring indicated in can be replaced with a series of rings of
appropriate cross-sectional shape.

[0103] Reservoirs and wells used for loading one or more samples onto the
microfluidic device of the present invention include but are not limited
to pipettes, syringes, cartridges, vials, eppendorf tubes and cell
culture materials (e.g., 96 well plates) as described above. One of the
issues to be resolved in loading samples into the inlet channel at the
inlet module of the substrate is the size difference between the loading
means or injection means, e.g., capillary or HPLC tubing and the inlet
channel. It is necessary to create an interconnect and loading method
which limits leaks and minimizes dead volume and compliance problems.
Several devices and methods described in further detail herein address
and solve these art problems.

Self-Aligning Fluidic Interconnects

[0104] The present invention includes one or more inlet modules comprising
self-aligning fluidic interconnects proximate to one or more inlet
channels to improve the efficiency of sample loading and/or injection.

[0105] The present invention proposes the use of small interconnects based
on creating a radial seal instead of a face seal between the microfluidic
device and interconnect. The inserted interconnect would have a larger
diameter than the mating feature on the device. When inserted, the
stretching of the chip would provide the sealing force needed to make a
leak-free seal between the external fluid lines and the microfluidic
device. FIG. 17 details design possibilities for making this seal.

[0106] Studies were performed using a cast hole in PDMS and 1/32'' PEEK
tubing, which showed that the seal was able to withstand more than 90 PSI
of pressure without leakage.

[0107] In order to handle instrument and chip manufacturing tolerances,
the external interconnect must be self-aligning and the "capture radius"
of the molded hole must be large enough to reliably steer the
interconnect to the sealing surfaces. FIG. 18 shows that the entrance to
the molded hole is large enough to guarantee capture but tapers down to
the sealing surfaces. The external interconnect could be made directly
out of the tubing leading up to the microfluidic substrate, thus
eliminating potential leak points and unswept volumes. As seen in FIG.
18, the interconnect is surrounded by the substrate interconnects or
"chip dock" for most of its length to make certain it is held within the
tolerance stack-up of the system. The external interconnect is made from
a hard but flexible material such as 1/32'' PEEK tubing. The features in
the microfluidic device can be molded directly into it during the
manufacturing process, while the inserted seals can be molded/machined
directly onto the tubing ends or molded as individual pieces and
mechanically fastened to the tubing. The retaining ferrule shown in FIG.
18 would be attached during manufacturing and provide good absolute
referencing of the tube length. The ferrule could be an off-the-shelf
component or a custom manufactured part and be made from, for example, a
polymer, an elastomer, or a metal. The tubing end could be tapered on the
end (top most diagram) or squared off (the figure above). The specific
shape of the end will be controlled by how easily the microfluidic device
will gall during insertion.

[0108] Alternatively, it is also possible to mold all the interconnects
needed for each tube into a single monolithic self-aligned part as
detailed in FIG. 19. This may help reduce the difficulty in maintaining
alignment of many external fluidic lines to the chip.

Elastomeric Fluid Interconnects

[0109] A conceptual layout of a microfluidic chip having an elastomeric
radial seal (also referred to herein as a "gasket") interface between the
fluidic plate and a means for introducing a sample (e.g., a pipette or
tubing) is shown in FIG. 1. A cross section of the microfluidic chip
depicted in FIG. 1 is shown in FIG. 2. As shown in FIG. 2, the fluidic
plate contains one or more port structures that include a tapered lead
directly into a microfluidic channel. The elastomeric gasket includes one
or more tapered bosses that are configured to fit within the one or more
port structures in the microfluidic chip. The gasket and fluidic plate
depicted in FIG. 2 are shown assembled in FIG. 3. As shown in FIG. 4, the
downward force of the sample introduction means (also referred to herein
as a "fluid transport mechanism", e.g., a pipette or tubing) radially
compresses (Z force) the gasket, thereby creating a seal between the
gasket and the port structure in the microfluidic chip. The gasket can be
loosely aligned with the one or more port structures prior to sealing by
the radial compression applied by the sample introduction means.
Optionally, the gasket/chip assembly can be staked (e.g., heat bonded,
glued or clamped) to a carrier apparatus prior to sealing to facilitate
insertion of the assembly into an instrument for analysis. Staking of the
gasket/chip assembly to a carrier causes axial compression against the
gasket to further induce sealing between the gasket and port assembly.
However, axial compression is not required. The radial compression by the
sample introduction means is sufficient to seal the gasket.

[0110] The conceptual design depicted in FIGS. 1-4 minimizes the
requirements on precision of the fluid interface, and can accommodate
many options for materials of different durometer. Although the
conceptual design depicted in FIGS. 1-4 requires an additional mold to
produce the gasket, and post-mold assembly with the fluid plate,
assembly/alignment of the loose parts is not expected to add any
significant complexity to the assembly. Furthermore, the design keeps a
planar part for ease of bonding and creates all disposable wetted parts
to eliminate any cross contamination.

[0111] Shifting focus now to the port modules or ports within the
microfluidic chip, the port modules can be configured to accommodate a
variety of different shapes and sizes of different types of sample
introduction means. For example, the port module within the gasket can be
designed to accommodate tubing (e.g., PEEK tubing), a 10 μL pipette, a
25 μL pipette, a 50 μL pipette, a 100 μL pipette, a 500 μL
pipette, a 1000 μL pipette, etc. Six exemplary embodiments of
different configurations for the port modules within the fluidic plate
are depicted in FIG. 5 (depicted as Detail C through Detail H). As shown
in FIG. 5, the dimensions and angle of the port module can vary. For
example, the port module can be substantially perpendicular to the
microfluidic channel, as depicted in Detail E in FIG. 5. Alternatively,
the port module can include a tapered angle of varying degrees, as
depicted in Detail C, D, F, G, and H.

[0112] It should be noted that a portion of the gasket is configured to
fit at least partially into a port, while another portion of the gasket
is configured to sealingly receive the pipette or other means for
introducing a sample fluid (e.g., tubing). In particular, a bottom
portion of the tapered bosses formed within the gasket are configured to
align and fit at least partially within the port modules in the fluid
plate. A top portion of the same bosses receive the means for introducing
a sample fluid (e.g., a tube or pipette). As such, the bosses within the
gasket should be of similar dimensions and angles as the port modules
with which they are aligned.

[0113] In certain embodiments, the microfluidic chip/gasket assembly is
housed within a plastic carrier. A plastic housing can be useful for
stacking the microfluidic chips within an instrument, particularly a
robotic instrument. The plastic carrier can include information such as a
bar code to identify particular sample fluids and/or experiments being
conducted within the microfluidic chip. Alternatively, a bar code can be
printed directly on the microfluidic chip.

[0114] Where a plastic carrier is used, the chip/gasket assembly can be
held within the plastic carrier by a clamp, or can be heat-staked or
glued to the plastic carrier. Clamping, heat-staking or gluing the
chip/gasket assembly to the plastic carrier provides axial compression
against the gasket to help induce a fluid-tight seal at the fluid
interface, in addition to the radial compression provided against the
gasket by insertion of a sample introduction means into a port module.
However, it should be noted that axial compression against the gasket is
not necessary to induce a fluid-tight seal at the fluid interface. A
sufficiently strong seal (e.g., able to hold pressure up to 100 psi) can
be created by radial compression only against the gasket.

[0115] The microfluidic chip/gasket/plastic carrier can be assembled in a
variety of configurations. Exemplary embodiments of the different
configurations are described in Table 1 below.

[0116] In Configuration 1, the microfluidic chip and gaskets are injection
molded separately and assembled within a 2 piece or 1 piece plastic
carrier, depending on whether a clamp is used to fix the chip/gasket
assembly within the plastic carrier (i.e., a 2 piece carrier). The
microfluidic chip includes a top plate and a bottom plate that are bonded
together. The top and bottom plates are of uniform thickness (e.g., 1.7
mm). The bottom plate has microfluidic channels molded or etched into the
plate. The top plate includes port modules that lead directly into the
microfluidic channels when the top plate is fitted over the bottom plate.
The gasket is fitted over the top plate, the bosses being aligned with
the port modules in the top plate. The chip/gasket assembly is inserted
into a plastic carrier. A clamp can be used to fix the chip to the
carrier (2 piece carrier) and provides axial compression against the
gasket interface. Alternatively the chip can be heat-staked or glued to
the plastic carrier (1 piece carrier).

[0117] In Configuration 2, the microfluidic chip includes a top plate and
a bottom plate that are bonded together, as described in Configuration 1.
However, the gasket is overmolded directly onto the top plate of the
microfluidic chip (see. for example, FIGS. 6A and 6B), instead of being
separately injection molded. As such, no separate chip/gasket assembly
step is required. The chip/gasket assembly is then fixed to a plastic
carrier by a clamp (2 piece carrier), or by heat-staking or gluing (1
piece carrier).

[0118] In Configuration 3, the microfluidic chip includes a top plate and
a bottom plate, as described in Configuration 1. The top plate has
pockets for gaskets to be molded into it. The gaskets are placed into the
top plate prior to bonding the top and bottom plate together, for example
by heat sealing (see FIGS. 7A-7D). In this configuration, the gasket is
contained within the chip, and the carrier features are designed into the
top and bottom plate. In other words, the chip itself is the carrier. A
separate plastic carrier is not necessary. As such, Configuration 3 has
an overall decreased thickness as compared to Configurations which
utilize a plastic carrier, such as those described in Configurations 1
and 2. Other features can be designed into the chip to protect sensitive
areas, such as an imaging FOV.

[0119] Configuration 4 is similar to Configuration 3, except that the
chip/gasket assembly is fixed to a plastic carrier.

[0120] The gasket can be made of a variety of materials of different
durometers. Preferably, the gasket is made of a material that is
compatible for use with water and oil-based fluids, and in particular, a
fluorinated oil. Suitable materials include elastomeric materials having
a hardness, shore A ranging from 20.0-75.0, preferably 30.0-60.0, more
preferably 40.0 to 55.0, a processing temperature ranging from
300° F. to 500° F., a feed temperature of about
80°-100° F., a mold temperature ranging from 60° F.
to 105° F., and an injection pressure ranging from 250 psi to 7500
psi. In particular embodiments, the gasket is made of a thermoplastic
silicone elastomer, such as Geniomer® 200 Silicone TPE (Wacker
Chemie), which is a two phase block copolymer made up of a soft
polydimethylsiloxane (PDMS) phase and a hard aliphatic isocyanate phase.
On account of its structure and the high siloxane content (over 90%), the
material is highly flexible and combines excellent transparency with good
mechanical properties. Geniomer® 200 contains neither plasticizers
nor reinforcing fillers. It can be processed using standard thermoplastic
processing techniques, and is particularly suitable for injection molding
because of its low melt viscosity. Such materials are capable resisting
flaking and degradation in the presence of a fluorinated oil, and/or
after sealingly receiving a means for introducing a sample fluid (e.g., a
tubing or pipette)

[0121] The microfluidic chip can be injection molded from a variety of
materials. Preferably the microfluidic chip is injection molded using a
cyclic olefin copolymer (COC).

[0122] The microfluidic chip and gasket interface can be injection molded
as individual components that are assembled together. Alternatively, the
gasket interface can be overmolded directly onto the fluidic plate. For
example, the gasket interface can be overmolded onto the entire surface
of the fluid plate, with tapered bosses aligned with the port modules
within the fluid plate, or the gasket interface can be overmolded within
each individual port module within the fluid plate.

[0123] The plastic carrier and clamp can also be injection molded from a
variety of materials. Preferably, the plastic carrier and clamp are
injection molded using acrylonitrile butadiene styrene (ABS).

[0124] A preferred embodiment of a gasket interface for use in a
microfluidic chip is depicted in FIG. 8. In this particular embodiment,
the gasket is injection molded using Genomier® 200. The gasket is
then assembled to a microfluidic chip having three port modules which
align with the bosses on the gasket. FIGS. 9A and 9B depict the preferred
embodiment of the fluid interface with a microfluidic chip using the
gasket depicted in FIG. 8 is depicted in FIGS. 9A and 9B. As shown in
FIGS. 9A and 9B, the gasket/chip assembly is configured to accommodate a
variety of means for introducing a sample fluid, including PEEK tubing, a
50 uL pipette, and a 1 mL pipette. It should be noted that one or more of
the gaskets depicted in FIG. 8 can be assembled with a microfluidic chip,
so long as the chip has an appropriate number of corresponding port
structures to align with the bosses on the gaskets.

[0125] FIGS. 10-16 depict different perspective views of a preferred
embodiment of a full gasket/chip/carrier assembly, which includes the
gasket and the fluid interface depicted in FIGS. 8 and 9, respectively.
The device depicted in FIGS. 10-16 further include a member defining at
least three internal channels, each channel having an inlet port and an
outlet port. The member includes a top plate adhered to a bottom plate,
where each of the top and bottom plates has a top surface and a bottom
surface, and where the top surface of the bottom plate faces and is
adhered to the bottom surface of the top plate. The bottom plate defines
the channels, while the top plate defines the ports.

[0126] In FIGS. 10-16, a first gasket is associated with the first,
second, and third inlet ports and is configured to sealingly receive an
input pipette or tubing such that fluid exits a tip of the input pipette
or tubing and enters one of the first, second, and third channels via one
of the first, second, and third inlet ports. A second gasket associated
with the first, second, and third outlet ports is configured to sealingly
receive an output pipette or tubing such that fluid exits one of the
first, second, and third channels via one of the first, second, and third
outlet ports and enters a tip of the output pipette or tubing. The first
and second gaskets are each injection molded from a thermoplastic
silicone elastomer, such as Genomier® 200.

[0127] The first gasket includes a first bottom portion that fits at least
partially into the first inlet port, a second bottom portion that fits at
least partially into the second inlet port, and a third bottom portion
that fits at least partially into the third inlet port. The first gasket
further includes a first, second and third top portion that sealingly
receives the input pipette to allow fluid that exits the tip of the input
pipette to enter the first, second and third channels, respectively.

[0128] The second gasket includes a first, a second and a third bottom
portion that fits at least partially into the first, second and third
outlet ports, respectively. The second gasket further includes a first, a
second and a third top portion that sealingly receives the output pipette
or tube to allow fluid that exits the first, second and third channels to
enter the output pipette or tube.

[0129] It is noted that the assembly depicted in FIGS. 10-16 corresponds
to Configuration 1 described in Table 1 above, using a 2 piece plastic
carrier that includes a bar code label as a means for identifying the
sample fluid and/or experiment being conducted within the microfluidic
chip.

[0130] A disposable cartridge for use with a microfluidic analysis system
is also provided herein. The disposable cartridge includes a carrier and
a microfluidic device disposed within the carrier, such as the
microfluidic device described and depicted in FIGS. 10-16. For example,
the microfluidic device includes a member defining at least three
internal channels and also defining a first inlet port and a first outlet
port of a first one of the channels, a second inlet port and a second
outlet port of a second one of the channels, and a third inlet port and a
third outlet port of a third one of the channels. A first gasket is
associated with the first, second, and third inlet ports and configured
to sealingly receive an input pipette such that fluid exits a tip of the
input pipette and enters one of the first, second, and third channels via
one of the first, second, and third inlet ports. A second gasket
associated with the first, second, and third outlet ports and configured
to sealingly receive an output pipette such that fluid exits one of the
first, second, and third channels via one of the first, second, and third
outlet ports and enters a tip of the output pipette.

[0131] Microfluidic chips are generally designed as a single-use,
disposable chip, to avoid cross-contamination in biological, chemical and
diagnostic assays. The gasket interfaces described herein can be
disposable with the chip to avoid cross-contamination. Unlike previous
fluid interface designs for pressure-driven microfluidic systems in which
manufacturing of the interface can be complicated and expensive (e.g.,
Luer-Loc systems in which connection requires a twisting motion), the
elastomeric gaskets described herein can be injection molded and are
easily assembled with a microfluidic chip, or can be overmolded directly
onto the microfluidic chip.

Methods for Molding Fluidic Interconnects Directly on the Substrate

[0132] The present invention also provides methods of direct molding of
fluidic interconnects into a microfluidic device. Development of a
commercial microfluidic platform requires a simple, reliable fluidic
interconnect in order to reduce the chance of operator error and leaks.
Molding these interconnects directly into the microfluidic device
requires precise alignment of the molding pins to the patterned shim (the
"master" manufactured from Silicon/photoresist or made from some metal)
used to form the microfluidic and electrical channels. The extreme
tolerances required when molding with a low viscosity elastomer such as
PDMS requires near perfect sealing of the pin face to the master, while
still accommodating imperfections in the master and assembly of the
molding tool. In an embodiment, the present invention provides a precise
and repeatable method of molding of interconnects while accommodating the
imperfections in the molding process by introducing movable pins captured
in an elastomeric sleeve molded directly into the tool. In order to
effectively produce at relatively low volume and be able to inexpensively
prototype devices, the tool must be able to use masters generated using
standard photolithographic processes (e.g. silicon wafers patterned with
SU-8).

[0133]FIG. 20 shows a schematic of a molding tool based on this concept.
In FIG. 20, the pins are captured within an elastomeric molded sleeve. A
compression plate made from a rigid backer plate and foam rubber is used
to apply gentle even pressure to the pins and generate the force needed
to make the pins uniformly contact the master. The molded sleeve was
found to be necessary to consistently prevent the uncured elastomer from
penetrating the region between the pin and the top plate. Early designs
used pins captured in tight clearance holes, and the pins would
frequently bind in place (even with lubricant), preventing smooth motion
of the pins and improper contact with the master. This would in turn
cause a thin film of the elastomer to form between the bottom of the pin
and the master ("Flash"). This flash prevents proper operation of the
interconnects during chip operation. The addition of the elastomeric
sleeves around each pin eliminated this problem, and produce consistent,
reliable shutoff between the master and the pins.

Electrodes

[0134] The device can include channels for use in fluid control and other
channels filled with a metal alloy for casting integrated metal alloy
components (i.e., electrodes). Alternatively, the electrodes can be
manufactured using other technologies (e.g., lithographically patterned
electrodes made from indium tin oxide or a metal such as platinum). The
microfluidic device can include metal alloy components useful for
performing electrical functions on fluids, including but not limited to,
coalescing droplets, charging droplets, sorting droplets, detecting
droplets and shaking droplets to mix the contents of coalesced droplets.
The device can contain more than one of the above mentioned components
for more than one of the above mentioned functions.

[0135] The electrodes comprising metal alloy components may either
terminate at fluid channels or be isolated from fluid channels. The
electrodes can be constructed by filling the appropriate channels with
metal alloy. One way this can be accomplished is to use positive pressure
injection of the metal alloy in a melted state, such as with a syringe,
into the channels, and then cool the metal alloy to a solid form. Another
example is to use negative pressure to draw the metal alloy in a melted
state into the channels, and then cool the metal alloy to a solid form.
This can be accomplished for example by use of capillary forces. Another
method of construction can use any of the above mentioned embodiments,
and then flush out the metal alloy in a melted state with another liquid
to define the geometry of the metal alloy components. Another example is
to use any of the above mentioned embodiments, and then use a localized
cold probe to define a solid termination point for the metal alloy, and
then cool the remaining metal alloy to a solid form. A further example is
to use another material, such as microscopic solder spheres or UV curable
conductive ink, to form a barrier between fluid and metal alloy channels,
to define the geometry of the metal alloy components.

[0136] The device can include a combination of both integrated metal alloy
components and a patterned electrically conductive layer. The patterned
electrically conductive layer can have features patterned such that their
boundaries are within a leak-proof seal. The device can have a patterned
electrically conductive feature as one of two charging electrodes and one
integrated metal alloy component as the other of two charging electrodes.

[0137] The device can include a plurality of electrodes that are insulated
from the fluid present in the device, and the method of operation
including appropriate application of dielectrical signals and appropriate
fluids. In known devices, the electrodes are typically in contact with
the fluids in order to allow discharge of species that would otherwise
screen the applied dielectric field. Whereas, in devices where the
electrodes have been insulated from the fluid, this screening effect
typically arises so quickly that the device is not useful for any
significantly extended period of time. The drawbacks of electrodes in
contact with the fluids vs. insulated electrodes are (a) degraded
reliability against leaking (since the interface between the electrodes
and the other components of the device may be more difficult to effect a
leak-proof seal), and (b) degraded reliability against electrode
corrosion (whose failure mode effects include failure of application of
dielectric fields, and fluid channel contamination).

[0138] The device of the present invention comprising a plurality of
electrodes that are insulated from the fluid present in the device
counteracts this screening effect by extending the screening rise time
and including a polarity switch for all of the different dielectric
fields applied in the device. The screening rise time is extended by
using fluids with dielectrical properties. A polarity switch for all of
the different dielectric fields applied in the device is achieved by
using an algorithm for dielectrical control, which switches the polarity
of the dielectrical fields at a frequency sufficiently high to maintain
proper dielectrical function of the device. This dielectrical control
algorithm may also switch the polarity for the dielectric fields in a
cascading, time controlled manner starting at the fluid origin point and
progressing downstream, so that given fluid components experience one
polarity at every point along their course. The device of the present
invention can be used with metal alloy electrodes or using a combination
of metal alloy electrodes and patterned conductive film electrodes.

[0139] The invention can provide a microfluidic device using injected
electrodes. The interface between the microscopic electrode (typically 25
μm thick) and the macroscopic interconnect can easily fail if the
joint between the two is flexed. The flexing of the joint can be
eliminated by securing a firm material that serves to fasten, support,
and re-enforce the joint (i.e., a grommet) into the interface. In order
to prevent flexing, the mating surface of the device can be manufactured
from a hard material such as glass or plastic. The electrical connection
with the external system can be made by securing the device such that it
connects to a spring loaded contact, which is either offset from the
grommet (thereby minimizing the force applied to the solder region), or
centered on the grommet (as long as the contact does not touch the
solder).

[0140] The metal alloy components are also useful for performing optical
functions on fluids, including but not limited to, optical detection of
droplets in a geometry which may include a mirror.

[0141] To prevent leakage of fluid out of electrodes placed within
microfluidic channels, the microfluidic device can include a layer
patterned with channels for fluid control, and another layer with
patterned electrically conductive features, where the features are
patterned such that their boundaries are within a leak-proof seal. The
leak-proof seal can be achieved at the interface between the unpatterned
areas of the fluid control layer and the unpatterned areas of the
electrically conductive layer. The leak-proof seal can also be achieved
by a third interfacial layer between the fluid control layer and the
unpatterned areas of the electrically conductive layer. The third
interfacial layer can or cannot be perforated at specific locations to
allow contact between the fluid and the electrically conductive layer.
Electrical access ports can also be patterned in the fluid control layer.

[0142] The electrodes and patterned electrically conductive layers as
described can be associated with any module of the device as described
herein to generate dielectric or electric forces to manipulate and
control the droplets and their contents.

[0143] Effective control of uncharged droplets within microfluidic devices
can require the generation of extremely strong dielectric field
gradients. The fringe fields from the edges of a parallel plate capacitor
can provide an excellent topology to form these gradients. The
microfluidic device according to the present invention can include
placing a fluidic channel between two parallel electrodes, which can
result in a steep electric field gradient at the entrance to the
electrodes due to edge effects at the ends of the electrode pair. Placing
these pairs of electrodes at a symmetric channel split can allow precise
bi-directional control of droplet within a device. Using the same
principle, only with asymmetric splits, can allow single ended control of
the droplet direction in the same manner. Alternatively, a variation on
this geometry will allow precise control of the droplet phase by
shifting.

[0144] In some cases, transparent or substantially transparent electrodes
can be used. The electric field generator can be constructed and arranged
(e.g., positioned) to create an electric field applicable to the fluid of
at least about 0.01 V/micrometer, and, in some cases, at least about 0.03
V/micrometer, at least about 0.05 V/micrometer, at least about 0.08
V/micrometer, at least about 0.1 V/micrometer, at least about 0.3
V/micrometer, at least about 0.5 V/micrometer, at least about 0.7
V/micrometer, at least about 1 V/micrometer, at least about 1.2
V/micrometer, at least about 1.4 V/micrometer, at least about 1.6
V/micrometer, or at least about 2 V/micrometer. In some embodiments, even
higher electric field intensities may be used, for example, at least
about 2 V/micrometer, at least about 3 V/micrometer, at least about 5
V/micrometer, at least about 7 V/micrometer, or at least about 10
V/micrometer or more.

[0145] As described, an electric field may be applied to fluidic droplets
to cause the droplets to experience an electric force. The electric force
exerted on the fluidic droplets may be, in some cases, at least about
10-16 N/μm3. In certain cases, the electric force exerted on
the fluidic droplets may be greater, e.g., at least about 10-15
N/μm3, at least about 10-14 N/μm3, at least about
10-13 N/μm3, at least about 10-12 N/μm3, at
least about 10-11 N/μm3, at least about
10-10N/μm3, at least about 10-9N/μm3, at least
about 10-8 N/μm3, or at least about 10-7 N/μm3
or more. The electric force exerted on the fluidic droplets, relative to
the surface area of the fluid, may be at least about 10-15
N/μm2, and in some cases, at least about 10-14
N/μm2, at least about 10-13 N/μm2, at least about
10-12 N/μm2, at least about 10-11 N/μm2, at
least about 10-10 N/μm2, at least about 10-9
N/μm2, at least about 10-8 N/μm2, at least about
10-7 N/μm2, or at least about 10-6 N/μm2 or
more. In yet other embodiments, the electric force exerted on the fluidic
droplets may be at least about 10-9N, at least about 10-8N, at
least about 10-7N, at least about 10-6 N, at least about
10-5N, or at least about 10-4N or more in some cases.

Coalescence Module

[0146] The microfluidic device of the present invention also includes one
or more coalescence modules. A "coalescence module" is within or
coincident with at least a portion of the main channel at or downstream
of the inlet module where molecules, cells, small molecules or particles
comprised within droplets are brought within proximity of other droplets
comprising molecules, cells, small molecules or particles and where the
droplets in proximity fuse, coalesce or combine their contents. The
coalescence module can also include an apparatus, for generating an
electric force.

[0147] The electric force exerted on the fluidic droplet may be large
enough to cause the droplet to move within the liquid. In some cases, the
electric force exerted on the fluidic droplet may be used to direct a
desired motion of the droplet within the liquid, for example, to or
within a channel or a microfluidic channel (e.g., as further described
herein), etc.

[0148] The electric field can be generated from an electric field
generator, i.e., a device or system able to create an electric field that
can be applied to the fluid. The electric field generator may produce an
AC field (i.e., one that varies periodically with respect to time, for
example, sinusoidally, sawtooth, square, etc.), a DC field (i.e., one
that is constant with respect to time), a pulsed field, etc. The electric
field generator may be constructed and arranged to create an electric
field within a fluid contained within a channel or a microfluidic
channel. The electric field generator may be integral to or separate from
the fluidic system containing the channel or microfluidic channel,
according to some embodiments. As used herein, "integral" means that
portions of the components integral to each other are joined in such a
way that the components cannot be in manually separated from each other
without cutting or breaking at least one of the components.

[0149] Techniques for producing a suitable electric field (which may be
AC, DC, etc.) are known to those of ordinary skill in the art. For
example, in one embodiment, an electric field is produced by applying
voltage across a pair of electrodes, which may be positioned on or
embedded within the fluidic system (for example, within a substrate
defining the channel or microfluidic channel), and/or positioned
proximate the fluid such that at least a portion of the electric field
interacts with the fluid. The electrodes can be fashioned from any
suitable electrode material or materials known to those of ordinary skill
in the art, including, but not limited to, silver, gold, copper, carbon,
platinum, copper, tungsten, tin, cadmium, nickel, indium tin oxide
("ITO"), etc., as well as combinations thereof.

Detection Module

[0150] The microfluidic device of the present invention can also include
one or more detection modules. A "detection module" is a location within
the device, typically within the main channel where molecules, cells,
small molecules or particles are to be detected, identified, measured or
interrogated on the basis of at least one predetermined characteristic.
The molecules, cells, small molecules or particles can be examined one at
a time, and the characteristic is detected or measured optically, for
example, by testing for the presence or amount of a reporter. For
example, the detection module is in communication with one or more
detection apparatuses. The detection apparatuses can be optical or
electrical detectors or combinations thereof. Examples of suitable
detection apparatuses include optical waveguides, microscopes, diodes,
light stimulating devices, (e.g., lasers), photo multiplier tubes, and
processors (e.g., computers and software), and combinations thereof,
which cooperate to detect a signal representative of a characteristic,
marker, or reporter, and to determine and direct the measurement or the
sorting action at the sorting module. However, other detection techniques
can also be employed

[0151] The term "determining," as used herein, generally refers to the
analysis or measurement of a species, for example, quantitatively or
qualitatively, and/or the detection of the presence or absence of the
species. "Determining" may also refer to the analysis or measurement of
an interaction between two or more species, for example, quantitatively
or qualitatively, or by detecting the presence or absence of the
interaction. Examples of suitable techniques include, but are not limited
to, spectroscopy such as infrared, absorption, fluorescence, UV/visible,
FTIR ("Fourier Transform Infrared Spectroscopy"), or Raman; gravimetric
techniques; ellipsometry; piezoelectric measurements; immunoassays;
electrochemical measurements; optical measurements such as optical
density measurements; circular dichroism; light scattering measurements
such as quasielectric light scattering; polarimetry; refractometry; or
turbidity measurements as described further herein.

[0152] A detection module is within, communicating or coincident with a
portion of the main channel at or downstream of the inlet module and, in
sorting embodiments, at, proximate to, or upstream of, the sorting module
or branch point. The sorting module may be located immediately downstream
of the detection module or it may be separated by a suitable distance
consistent with the size of the molecules, the channel dimensions and the
detection system. Precise boundaries for the detection module are not
required, but are preferred.

[0153] Detection modules used for detecting molecules and cells have a
cross-sectional area large enough to allow a desired molecule, cells,
bead, or particles to pass through without being substantially slowed
down relative to the flow carrying it. The dimensions of the detection
module are influenced by the nature of the sample under study and, in
particular, by the size of the droplets, beads, particles, molecules or
cells (including virions) under study. For example, mammalian cells can
have a diameter of about 1 to 50 microns, more typically 10 to 30
microns, although some mammalian cells (e.g., fat cells) can be larger
than 120 microns. Plant cells are generally 10 to 100 microns. However,
other molecules or particles can be smaller with a diameter from about 20
nm to about 500 nm.

Mixing Module

[0154] The microfluidic device of the present invention can further
include one or more mixing modules. Although coalescence of one or more
droplets in one or more coalescence modules can be sufficient to mix the
contents of the coalesced droplets (e.g., through rotating vortexes
existing within the droplet), it should be noted that when two droplets
fuse or coalesce, perfect mixing within the droplet does not
instantaneously occur. Instead, for example, the coalesced droplet may
initially be formed of a first fluid region (from the first droplet) and
a second fluid region (from the second droplet). Thus, in some cases, the
fluid regions may remain as separate regions, for example, due to
internal "counter-revolutionary" flow within the fluidic droplet, thus
resulting in a non-uniform fluidic droplet. A "mixing module" can
comprise features for shaking or otherwise manipulate droplets so as to
mix their contents. The mixing module is preferably downstream from the
coalescing module and upstream from the detection module. The mixing
module can include, but is not limited to, the use of channel geometries,
acoustic actuators, metal alloy component electrodes or electrically
conductive patterned electrodes to mix the contents of droplets and to
reduce mixing times for fluids combined into a single droplet in the
microfluidic device. For example, the fluidic droplet may be passed
through one or more channels or other systems which cause the droplet to
change its velocity and/or direction of movement. The change of direction
may alter convection patterns within the droplet, causing the fluids to
be at least partially mixed. Combinations are also possible.

[0155] For acoustic manipulation, the frequency of the acoustic wave
should be fine-tuned so as not to cause any damage to the cells. The
biological effects of acoustic mixing have been well studied (e.g., in
the ink-jet industry) and many published literatures also showed that
piezoelectric microfluidic device can deliver intact biological payloads
such as live microorganisms and DNA. In an example, the design of the
acoustic resonant uses a Piezoelectric bimorph flat plate located on the
side of the carved resonant in the PDMS slab. The piezoelectric driving
waveform is carefully optimized to select the critical frequencies that
can separate cells in fluids. There are five parameters to optimize
beyond the frequency parameter. Lab electronics is used to optimize the
piezoelectric driving waveform. Afterwards, a low cost circuit can be
designed to generate only the optimized waveform in a preferred
microfluidic device.

[0156] The frequency of the acoustic wave should be fine-tuned so as not
to cause any damage to the cells. The biological effects of acoustic
mixing have been well studied (e.g., in the ink-jet industry) and many
published literatures also showed that piezoelectric microfluidic device
can deliver intact biological payloads such as live microorganisms and
DNA.

Sensors

[0157] One or more detections sensors and/or processors may be positioned
to be in sensing communication with the fluidic droplet. "Sensing
communication," as used herein, means that the sensor may be positioned
anywhere such that the fluidic droplet within the fluidic system (e.g.,
within a channel), and/or a portion of the fluidic system containing the
fluidic droplet may be sensed and/or determined in some fashion. For
example, the sensor may be in sensing communication with the fluidic
droplet and/or the portion of the fluidic system containing the fluidic
droplet fluidly, optically or visually, thermally, pneumatically,
electronically, or the like. The sensor can be positioned proximate the
fluidic system, for example, embedded within or integrally connected to a
wall of a channel, or positioned separately from the fluidic system but
with physical, electrical, and/or optical communication with the fluidic
system so as to be able to sense and/or determine the fluidic droplet
and/or a portion of the fluidic system containing the fluidic droplet
(e.g., a channel or a microchannel, a liquid containing the fluidic
droplet, etc.). For example, a sensor may be free of any physical
connection with a channel containing a droplet, but may be positioned so
as to detect electromagnetic radiation arising from the droplet or the
fluidic system, such as infrared, ultraviolet, or visible light. The
electromagnetic radiation may be produced by the droplet, and/or may
arise from other portions of the fluidic system (or externally of the
fluidic system) and interact with the fluidic droplet and/or the portion
of the fluidic system containing the fluidic droplet in such as a manner
as to indicate one or more characteristics of the fluidic droplet, for
example, through absorption, reflection, diffraction, refraction,
fluorescence, phosphorescence, changes in polarity, phase changes,
changes with respect to time, etc. As an example, a laser may be directed
towards the fluidic droplet and/or the liquid surrounding the fluidic
droplet, and the fluorescence of the fluidic droplet and/or the
surrounding liquid may be determined. "Sensing communication," as used
herein may also be direct or indirect. As an example, light from the
fluidic droplet may be directed to a sensor, or directed first through a
fiber optic system, a waveguide, etc., before being directed to a sensor.

[0158] Non-limiting examples of detection sensors useful in the invention
include optical or electromagnetically-based systems. For example, the
sensor may be a fluorescence sensor (e.g., stimulated by a laser), a
microscopy system (which may include a camera or other recording device),
or the like. As another example, the sensor may be an electronic sensor,
e.g., a sensor able to determine an electric field or other electrical
characteristic. For example, the sensor may detect capacitance,
inductance, etc., of a fluidic droplet and/or the portion of the fluidic
system containing the fluidic droplet. In some cases, the sensor may be
connected to a processor, which in turn, cause an operation to be
performed on the fluidic droplet, for example, by sorting the droplet.

Processors

[0159] As used herein, a "processor" or a "microprocessor" is any
component or device able to receive a signal from one or more sensors,
store the signal, and/or direct one or more responses (e.g., as described
above), for example, by using a mathematical formula or an electronic or
computational circuit. The signal may be any suitable signal indicative
of the environmental factor determined by the sensor, for example a
pneumatic signal, an electronic signal, an optical signal, a mechanical
signal, etc.

[0160] The device of the present invention can comprise features, such as
integrated metal alloy components and/or features patterned in an
electrically conductive layer, for detecting droplets by broadcasting a
signal around a droplet and picking up an electrical signal in proximity
to the droplet.

[0162] The terms used in this specification generally have their ordinary
meanings in the art, within the context of this invention and in the
specific context where each term is used. Certain terms are discussed
below, or elsewhere in the specification, to provide additional guidance
to the practitioner in describing the devices and methods of the
invention and how to make and use them. It will be appreciated that the
same thing can typically be described in more than one way. Consequently,
alternative language and synonyms may be used for any one or more of the
terms discussed herein. Synonyms for certain terms are provided. However,
a recital of one or more synonyms does not exclude the use of other
synonyms, nor is any special significance to be placed upon whether or
not a term is elaborated or discussed herein. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference. In the case of conflict, the present
specification, including definitions, will control. In addition, the
materials and methods described herein are illustrative only and are not
intended to be limiting.

[0163] As used herein, "about" or "approximately" shall generally mean
within 20 percent, preferably within 10 percent, and more preferably
within 5 percent of a given value or range.

[0164] The term "molecule" means any distinct or distinguishable
structural unit of matter comprising one or more atoms, and includes for
example polypeptides and polynucleotides.

[0165] The term "polymer" means any substance or compound that is composed
of two or more building blocks (`mers`) that are repetitively linked to
each other. For example, a "dimer" is a compound in which two building
blocks have been joined together.

[0166] The term "polynucleotide" as used herein refers to a polymeric
molecule having a backbone that supports bases capable of hydrogen
bonding to typical polynucleotides, where the polymer backbone presents
the bases in a manner to permit such hydrogen bonding in a sequence
specific fashion between the polymeric molecule and a typical
polynucleotide (e.g., single-stranded DNA). Such bases are typically
inosine, adenosine, guanosine, cytosine, uracil and thymidine. Polymeric
molecules include double and single stranded RNA and DNA, and backbone
modifications thereof, for example, methylphosphonate linkages.

[0167] Thus, a "polynucleotide" or "nucleotide sequence" is a series of
nucleotide bases (also called "nucleotides") generally in DNA and RNA,
and means any chain of two or more nucleotides. A nucleotide sequence
typically carries genetic information, including the information used by
cellular machinery to make proteins and enzymes. These terms include
double or single stranded genomic and cDNA, RNA, any synthetic and
genetically manipulated polynucleotide, and both sense and anti-sense
polynucleotide (although only sense stands are being represented herein).
This includes single- and double-stranded molecules, i.e., DNA-DNA,
DNA-RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (PNA)
formed by conjugating bases to an amino acid backbone. This also includes
nucleic acids containing modified bases, for example thio-uracil,
thio-guanine and fluoro-uracil.

[0168] The polynucleotides herein may be flanked by natural regulatory
sequences, or may be associated with heterologous sequences, including
promoters, enhancers, response elements, signal sequences,
polyadenylation sequences, introns, 5'- and 3'-non-coding regions, and
the like. The nucleic acids may also be modified by many means known in
the art. Non-limiting examples of such modifications include methylation,
"caps", substitution of one or more of the naturally occurring
nucleotides with an analog, and internucleotide modifications such as,
for example, those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged
linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
Polynucleotides may contain one or more additional covalently linked
moieties, such as, for example, proteins (e.g., nucleases, toxins,
antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g.,
acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals,
iron, oxidative metals, etc.), and alkylators. The polynucleotides may be
derivatized by formation of a methyl or ethyl phosphotriester or an alkyl
phosphoramidate linkage. Furthermore, the polynucleotides herein may also
be modified with a label capable of providing a detectable signal, either
directly or indirectly. Exemplary labels include radioisotopes,
fluorescent molecules, biotin, and the like.

[0169] The term "dielectrophoretic force gradient" means a
dielectrophoretic force is exerted on an object in an electric field
provided that the object has a different dielectric constant than the
surrounding media. This force can either pull the object into the region
of larger field or push it out of the region of larger field. The force
is attractive or repulsive depending respectively on whether the object
or the surrounding media has the larger dielectric constant.

[0170] "DNA" (deoxyribonucleic acid) means any chain or sequence of the
chemical building blocks adenine (A), guanine (G), cytosine (C) and
thymine (T), called nucleotide bases, that are linked together on a
deoxyribose sugar backbone. DNA can have one strand of nucleotide bases,
or two complimentary strands which may form a double helix structure.
"RNA" (ribonucleic acid) means any chain or sequence of the chemical
building blocks adenine (A), guanine (G), cytosine (C) and uracil (U),
called nucleotide bases, that are linked together on a ribose sugar
backbone. RNA typically has one strand of nucleotide bases.

[0171] A "polypeptide" (one or more peptides) is a chain of chemical
building blocks called amino acids that are linked together by chemical
bonds called peptide bonds. A "protein" is a polypeptide produced by a
living organism. A protein or polypeptide may be "native" or "wild-type",
meaning that it occurs in nature; or it may be a "mutant", "variant" or
"modified", meaning that it has been made, altered, derived, or is in
some way different or changed from a native protein, or from another
mutant.

[0172] As used herein, "particles" means any substance that may be
encapsulated within a droplet for analysis, reaction, sorting, or any
operation according to the invention. Particles are not only objects such
as microscopic beads (e.g., chromatographic and fluorescent beads),
latex, glass, silica or paramagnetic beads, but also includes other
encapsulating porous and/or biomaterials such as liposomes, vesicles and
other emulsions. Beads ranging in size from 0.1 micron to 1 mm can be
used in the devices and methods of the invention and are therefore
encompassed with the term "particle" as used herein. The term particle
also encompasses biological cells, as well as beads and other microscopic
objects of similar size (e.g., from about 0.1 to 120 microns, and
typically from about 1 to 50 microns) or smaller (e.g., from about 0.1 to
150 nm). The devices and methods of the invention are also directed to
sorting and/or analyzing molecules of any kind, including
polynucleotides, polypeptides and proteins (including enzymes) and their
substrates and small molecules (organic or inorganic). Thus, the term
particle further encompasses these materials.

[0173] The particles (including, e.g., cells and molecules) are sorted
and/or analyzed by encapsulating the particles into individual droplets
(e.g., droplets of aqueous solution in oil), and these droplets are then
sorted, combined and/or analyzed in a microfabricated device.
Accordingly, the term "droplet" generally includes anything that is or
can be contained within a droplet.

[0174] A "small molecule" as used herein, is meant to refer to a
composition that has a molecular weight of less than about 5 kD and most
preferably less than about 4 kD. Small molecules can be, e.g., nucleic
acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or
other organic or inorganic molecules. Libraries of chemical and/or
biological mixtures, such as fungal, bacterial, or algal extracts, are
known in the art.

[0175] As used herein, "cell" means any cell or cells, as well as viruses
or any other particles having a microscopic size, e.g. a size that is
similar to or smaller than that of a biological cell, and includes any
prokaryotic or eukaryotic cell, e.g., bacteria, fungi, plant and animal
cells. Cells are typically spherical, but can also be elongated,
flattened, deformable and asymmetrical, i.e., non-spherical. The size or
diameter of a cell typically ranges from about 0.1 to 120 microns, and
typically is from about 1 to 50 microns. A cell may be living or dead.
Since the microfabricated device of the invention is directed to sorting
materials having a size similar to a biological cell (e.g. about 0.1 to
120 microns) or smaller (e.g., about 0.1 to 150 nm) any material having a
size similar to or smaller than a biological cell can be characterized
and sorted using the microfabricated device of the invention. Thus, the
term cell shall further include microscopic beads (such as
chromatographic and fluorescent beads), liposomes, emulsions, or any
other encapsulating biomaterials and porous materials. Non-limiting
examples include latex, glass, or paramagnetic beads; and vesicles such
as emulsions and liposomes, and other porous materials such as silica
beads. Beads ranging in size from 0.1 micron to 1 mm can also be used,
for example in sorting a library of compounds produced by combinatorial
chemistry. As used herein, a cell may be charged or uncharged. For
example, charged beads may be used to facilitate flow or detection, or as
a reporter. Biological cells, living or dead, may be charged for example
by using a surfactant, such as SDS (sodium dodecyl sulfate). The term
cell further encompasses "virions", whether or not virions are expressly
mentioned.

[0176] A "virion", "virus particle" is the complete particle of a virus.
Viruses typically comprise a nucleic acid core (comprising DNA or RNA)
and, in certain viruses, a protein coat or "capsid". Certain viruses may
have an outer protein covering called an "envelope". A virion may be
either living (i.e., "viable") or dead (i.e., "non-viable"). A living or
"viable" virus is one capable of infecting a living cell. Viruses are
generally smaller than biological cells and typically range in size from
about 20-25 nm diameter or less (parvoviridae, picornoviridae) to
approximately 200-450 nm (poxyiridae). However, some filamentous viruses
may reach lengths of 2000 nm (closterviruses) and are therefore larger
than some bacterial cells. Since the microfabricated device of the
invention is particularly suited for sorting materials having a size
similar to a virus (i.e., about 0.1 to 150 nm), any material having a
size similar to a virion can be characterized and sorted using the
microfabricated device of the invention. Non-limiting examples include
latex, glass or paramagnetic beads; vesicles such as emulsions and
liposomes; and other porous materials such as silica beads. Beads ranging
in size from 0.1 to 150 nm can also be used, for example, in sorting a
library of compounds produced by combinatorial chemistry. As used herein,
a virion may be charged or uncharged. For example, charged beads may be
used to facilitate flow or detection, or as a reporter. Biological
viruses, whether viable or non-viable, may be charged, for example, by
using a surfactant, such as SDS.

[0177] A "reporter" is any molecule, or a portion thereof, that is
detectable, or measurable, for example, by optical detection. In
addition, the reporter associates with a molecule, cell or virion or with
a particular marker or characteristic of the molecule, cell or virion, or
is itself detectable to permit identification of the molecule, cell or
virion's, or the presence or absence of a characteristic of the molecule,
cell or virion. In the case of molecules such as polynucleotides such
characteristics include size, molecular weight, the presence or absence
of particular constituents or moieties (such as particular nucleotide
sequences or restrictions sites). In the case of cells, characteristics
which may be marked by a reporter includes antibodies, proteins and sugar
moieties, receptors, polynucleotides, and fragments thereof. The term
"label" can be used interchangeably with "reporter". The reporter is
typically a dye, fluorescent, ultraviolet, or chemiluminescent agent,
chromophore, or radio-label, any of which may be detected with or without
some kind of stimulatory event, e.g., fluoresce with or without a
reagent. In one embodiment, the reporter is a protein that is optically
detectable without a device, e.g. a laser, to stimulate the reporter,
such as horseradish peroxidase (HRP). A protein reporter can be expressed
in the cell that is to be detected, and such expression may be indicative
of the presence of the protein or it can indicate the presence of another
protein that may or may not be coexpressed with the reporter. A reporter
may also include any substance on or in a cell that causes a detectable
reaction, for example by acting as a starting material, reactant or a
catalyst for a reaction which produces a detectable product. Cells may be
sorted, for example, based on the presence of the substance, or on the
ability of the cell to produce the detectable product when the reporter
substance is provided.

[0178] A "marker" is a characteristic of a molecule, cell or virion that
is detectable or is made detectable by a reporter, or which may be
coexpressed with a reporter. For molecules, a marker can be particular
constituents or moieties, such as restrictions sites or particular
nucleic acid sequences in the case of polynucleotides. For cells and
virions, characteristics may include a protein, including enzyme,
receptor and ligand proteins, saccharides, polynucleotides, and
combinations thereof, or any biological material associated with a cell
or virion. The product of an enzymatic reaction may also be used as a
marker. The marker may be directly or indirectly associated with the
reporter or can itself be a reporter. Thus, a marker is generally a
distinguishing feature of a molecule, cell or virion, and a reporter is
generally an agent which directly or indirectly identifies or permits
measurement of a marker. These terms may, however, be used
interchangeably.

[0179] Certain embodiments according to the invention have been disclosed.
These embodiments are illustrative of, and not limiting on, the
invention. Other embodiments, as well as various modifications and
combinations of the disclosed embodiments, are possible and within the
scope of the disclosure.